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Journal: The Journal of pharmacology and experimental therapeutics


Dopaminergic neurons of the ventral tegmental area are important components of brain pathways related to addiction. Prolonged exposure of these neurons to moderate concentrations of dopamine (DA) decreases their sensitivity to inhibition by DA, a process called DA-inhibition reversal (DIR). DIR is mediated by phospholipase C and conventional subtype of protein kinase C (cPKC) through concurrent stimulation of D2 and D1-like DA receptors, or by D2 stimulation concurrent with activation of 5-HT(2) or neurotensin receptors. In the present study, we further characterized this phenomenon by use of extracellular recordings in brain slices to examine whether DIR is linked to G protein-coupled receptor kinase-2 (GRK2) or dynamin by assessing DIR in the presence of antagonists of these enzymes. DIR was blocked by β-ARK1 inhibitor, which inhibits GRK2, and by dynasore, which blocks dynamin. Reversal of inhibition by D2 agonist quinpirole was produced by serotonin (50 µM) and by neurotensin (5-10 nM). Serotonin-induced or neurotensin-induced reversal was blocked by β-ARK1 inhibitor, dynasore, or cPKC antagonist 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4c]carbazole-12-propanenitrile (Gö6976). This further characterization of DIR indicates that cPKC, GRK2, and dynamin play important roles in the desensitization of D2 receptors. As drugs of abuse produce persistent increases in DA concentration in the ventral tegmental area, reduction of D2 receptor sensitivity as a result of drug abuse may be a critical factor in the processes of addiction.

Concepts: Signal transduction, Enzyme inhibitor, Receptor antagonist, Dopamine receptor, Serotonin, Drug addiction, Addiction, Dopamine


Steatotic grafts are excluded for use in partial liver transplantation (LT) due to increased risk of primary non-function. This study investigated the effects of suramin, a polysulfonated naphthylurea, on the outcome of steatotic partial LT. Rat livers were harvested after acute ethanol treatment (6 g/kg, i.g.), reduced in size to ~1/3, and transplanted. Serum alanine aminotransferase (ALT) and total bilirubin, and hepatic necrosis and apoptosis were significantly higher after transplantation of fatty partial grafts (FPG) than lean partial grafts (LPG). Suramin (5 mg/kg, i.p.) decreased ALT by ~60%, hyperbilirubinemia by 75%, necrosis by 83%, and apoptosis by 70% after FPG transplantation. Hepatic cellular 5-bromo-2'-deoxyuridine (BrdU) incorporation increased to 28% in LPG but was only 2% in FPG at 48 h and mitotic index increased to 7% in LPG but was only 0.2% in FPG, indicating suppressed regeneration in FPG. Suramin increased BrdU incorporation and mitotic index to 43% and 9%, respectively, in FPG. All FPG recipients died within 5 days. Suramin recovered survival of FPG to 62%. TNFα mRNA was 2.2-fold higher in FPG than in LPG and was associated with activation of caspase-8 and -3 in FPG. Suramin decreased TNFα and caspase activation in FPG. TGF-β, phospho-Smad2/3 and p21Cip1 were significantly higher in FPG than in LPG and suramin blocked TGF-β formation and its down-stream signaling pathway. Taken together, suramin improves the outcome of FPG transplantation, most likely by inhibition of TNFα and TGF-β formation.

Concepts: Apoptosis, Cirrhosis, Liver, Liver function tests, Alanine transaminase, Hepatology, Bilirubin, Jaundice


In order to evaluate the relationship between the in vitro and in vivo potency of sodium-glucose cotransporter (SGLT) inhibitors, a pharmacokinetic and pharmacodynamic (PK-PD) study was performed using normal rats. A highly selective SGLT2 inhibitor, tofogliflozin, and four other inhibitors with different in vitro inhibition potency to SGLT2 and selectivity toward SGLT2 versus SGLT1 were used as test compounds, and the time courses for urinary glucose excretion (UGE) and the plasma glucose and compound concentrations were monitored after administration of the compounds. A PK-PD analysis of the UGE caused by SGLT inhibition was performed based on a nonlinear parallel tube model which took into consideration the consecutive reabsorption by different glucose transport properties of SGLT2 and SGLT1. The model adequately captured the time course of cumulative UGE caused by SGLT inhibition; then the in vivo inhibition constants (K(i)) of inhibitors for both SGLT1 and SGLT2 were estimated. The in vivo selectivity toward SGLT2 showed a good correlation with the in vitro data (r = 0.985, p < 0.05), with in vivo K(i) values for SGLT2 in the range of 0.3- to 3.4-fold the in vitro data. This suggests that in vitro inhibition potency to both SGLT2 and SGLT1 is reflected in vivo. Furthermore, the complementary role of SGLT1 to SGLT2 and how selectivity toward SGLT2 affects the inhibitory potency for renal glucose reabsorption were discussed using the PK-PD model.

Concepts: Inhibitor, Glucose transporter, Cotransporter, Co-transport, Robert K. Crane, Sodium-glucose transport proteins, SLC5A1, Symporter


JNJ-26854165 (serdemetan) has previously been reported to inhibit the function of the E3 ligase human double minute-2, and we initially sought to characterize its activity in models of mantle cell lymphoma (MCL) and multiple myeloma (MM). Serdemetan induced a dose dependent inhibition of proliferation in both wild-type (wt) and mutant (mut) p53 cell lines, with IC50’s from 0.25 μ/L to 3 μ/L, in association with an S phase cell cycle arrest. Caspase-3 activation was primarily seen in wtp53 bearing cells, but also occurred in mutp53 bearing cells, albeit to a lesser extent. 293T cells treated with JNJ-26854165, and serdemetan-resistant fibroblasts displayed accumulation of cholesterol within endosomes, a phenotype reminiscent of that seen in the ATP-binding cassette sub-family A member-1 (ABCA1) cholesterol transport disorder, Tangiers disease. MM and MCL cells had decreased cholesterol efflux and electron microscopy demonstrated the accumulation of lipid whorls, confirming the lysosomal storage disease phenotype. JNJ-26854165 induced induction of cholesterol regulatory genes sterol regulatory element-binding transcription factor-1 and -2, liver X receptors α and β, along with increased expression of Niemann-Pick disease type-C1 and -C2. However, JNJ-26854165 induced enhanced ABCA1 turnover despite enhancing transcription. Finally, ABCA1 depletion resulted in enhanced sensitivity to JNJ-26854165. Overall, these findings support the hypothesis that serdemetan functions in part by inhibiting cholesterol transport, and that this pathway is a potential new target for the treatment of MCL and MM.

Concepts: DNA, Protein, Gene, Golgi apparatus, Chromosome, Cell cycle, Proteasome, Mantle cell lymphoma


Chronic renal failure (CRF) is associated with the development of secondary hyperparathyroidism and vascular calcifications. We evaluated the efficacy of PA21, a new iron-based non-calcium phosphate binder, in controlling phosphocalcic disorders and preventing vascular calcifications in uremic rats. Rats with adenine-diet-induced CRF were randomized to receive either PA21 0.5%, 1.5% or 5% or calcium carbonate (CaCO3) 3% in the diet, for 4 weeks and were compared with uremic and non-uremic control groups. After 4 weeks' phosphate binder treatment, serum calcium, creatinine and body weight were similar between all CRF groups. Serum phosphorus was reduced with CaCO3 3% (2.06 mmol/l, P≤0.001), PA21 1.5% (2.29 mmol/l, P<0.05) and PA21 5% (2.21 mmol/l, P≤0.001) versus CRF controls (2.91 mmol/l). Intact parathyroid hormone was strongly reduced in the PA21 5% and CaCO3 3% CRF groups to a similar extent (1138 and 1299 pg/ml, respectively) versus CRF controls (3261 pg/ml, both P≤0.001). A lower serum fibroblast growth factor 23 concentration was observed in the PA21 5%, compared with CaCO3 3% and CRF, control groups. PA21 5% CRF rats had a lower vascular calcification score compared with CaCO3 3% CRF rats and CRF controls. In conclusion, PA21 was as effective as CaCO3 at controlling phosphocalcic disorders but superior in preventing the development of vascular calcifications in uremic rats. Thus, PA21 represents a possible alternative to calcium-based phosphate binders in CRF patients.

Concepts: Renal failure, Nephrology, Carbon dioxide, Parathyroid hormone, Calcium carbonate, Hyperparathyroidism, Organ failure, Phosphate binders


Alterations in expression patterns of alpha4beta2 nicotinic acetylcholine receptors (nAChR) have been demonstrated to alter cholinergic neurotransmission and implicated in neurological disorders including Autism, nicotine addiction, Alzheimer’s and Parkinson’s disease. Positive allosteric modulators (PAMs) represent promising new leads in the development of therapeutic agents for the treatment of these disorders. This study investigates the involvement of the beta2 containing subunit interfaces of alpha4beta2 receptors in the modulation of acetylcholine- (ACh) induced responses by the PAM desformylflustrabromine (dFBr). Eight amino acids on the principal face of the beta2 subunit were mutated to alanine in order to explore the involvement of this region in potentiation of ACh-induced currents by dFBr. ACh-induced responses obtained from wild type and mutant alpha4beta2 receptors expressed in Xenopus laevis oocytes were recorded in the presence and absence of dFBr using two-electrode voltage clamp electrophysiology. Wild type and mutant receptors were expressed in both high- (HS) and low- (LS) ACh sensitivity isoforms by using biased injection ratios of 1:5 or 5:1 alpha4: beta2 cRNA. Mutations were made in the B, C and A loops of the principal face of the beta2 subunit; regions not involved in binding of ACh. Mutant β2(Y120A) significantly eliminated dFBr potency in both isoform preparations. Several other mutations altered dFBr potentiation levels in both preparations. Our findings support the involvement of the principal face of the beta2 subunit in dFBr modulation of ACh-induced responses. Findings from this study will aid in the improved design of dFBr-like PAMs for potential therapeutic use.

Concepts: Nicotine, Neurotransmitter, Acetylcholine, Myasthenia gravis, Muscarinic acetylcholine receptor, Nicotinic acetylcholine receptor, Acetylcholine receptor, Alpha-4 beta-2 nicotinic receptor


According to the catecholaldehyde hypothesis, the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to the loss of nigrostriatal dopaminergic neurons in Parkinson’s disease. Monoamine oxidase-A (MAO-A) catalyzes the conversion of intra-neuronal dopamine to DOPAL and may serve as a therapeutic target. The “cheese effect” - paroxysmal hypertension evoked by tyramine-containing foodstuffs-limits clinical use of irreversible MAO-A inhibitors. Combined MAO-A/B inhibition decreases DOPAL production in rat pheochromocytoma PC12 cells, but whether reversible MAO-A inhibitors or MAO-B inhibitors decrease endogenous DOPAL production has been unknown. We compared the potencies of MAO inhibitors in attenuating DOPAL production and examined possible secondary effects on dopamine storage, constitutive release, synthesis, and auto-oxidation. Catechol concentrations were measured in cells and medium after incubation with the irreversible MAO-A inhibitor clorgyline, three reversible MAO-A inhibitors, or the MAO-B inhibitors selegiline or rasagiline for 180 minutes. Reversible MAO-A inhibitors were generally ineffective, whereas clorgyline (1 nM), rasagiline (500 nM), and selegiline (500 nM) decreased DOPAL levels in the cells and medium. All 3 drugs also increased dopamine and norepinephrine, decreased DOPA, and increased cysteinyl-dopamine concentrations in the medium, suggesting increased vesicular uptake and constitutive release, decreased dopamine synthesis, and increased dopamine auto-oxidation. In conclusion, clorgyline, rasagiline, and selegiline decrease production of endogenous DOPAL. At relatively high concentrations the latter drugs probably lose their selectivity for MAO-B. Possibly offsetting increased formation of potentially toxic oxidation products and decreased formation of DOPAL might account for the failure of large clinical trials of MAO-B inhibitors to demonstrate slowing of neurodegeneration in Parkinson’s disease.

Concepts: Parkinson's disease, Neurotransmitter, Epinephrine, Dopamine, Monoamine oxidase, Rasagiline, Phenethylamine, Monoamine oxidase inhibitors


Mephedrone (MEPH) is a β-ketoamphetamine stimulant drug of abuse that is often a constituent of illicit bath salts formulations. While MEPH bears remarkable similarities to methamphetamine (METH) in terms of chemical structure, as well as its neurochemical and behavioral effects, it has been shown to have a reduced neurotoxic profile compared to METH. The addition of a β-keto moiety and a 4-methyl ring substituent to METH yields MEPH, and a loss of direct neurotoxic potential. In the present study, 2 analogs of METH, methcathinone (MeCa) and 4-methylmethamphetamine (4MM), were assessed for their effects on mouse dopamine (DA) nerve endings to determine the relative contribution of each individual moiety to the loss of direct neurotoxicity in MEPH. Both MeCa and 4MM caused significant alterations in core body temperature as well as locomotor activity and stereotypy, but 4MM was found to elicit minimal dopaminergic toxicity only at the highest dose. By contrast, MeCa caused significant reductions in all markers of DA nerve ending damage over a range of doses. These results lead to the conclusion that ring substitution at the 4-position profoundly reduces the neurotoxicity of METH, whereas the β-keto group has much less influence on this property. While the mechanism(s) by which the 4-methyl substituent reduces METH-induced neurotoxicity remains unclear, it is speculated that this effect is mediated by a loss of DA-releasing action in MEPH and 4MM at the synaptic vesicle monoamine transporter, an effect that is thought to be critical for METH-induced neurotoxicity.

Concepts: Nervous system, Attention-deficit hyperactivity disorder, Nerve, Stimulant, Cocaine, Methamphetamine, Narcolepsy, Methcathinone


Cardiac glycosides have been used in the treatment of arrhythmias for more than 200 years. Two-pore-domain (K2P) potassium channels regulate cardiac action potential repolarization. Recently, K2P3.1 (TASK-1) has been implicated in atrial fibrillation (AF) pathophysiology and was suggested as atrial-selective antiarrhythmic drug target. We hypothesized that blockade of cardiac K2P channels contributes to the mechanism of action of digitoxin and digoxin. All functional human K2P channels were screened for interactions with cardiac glycosides. Human K2P channel subunits were expressed in Xenopus laevis oocytes, and voltage clamp electrophysiology was used to record K+ currents. Digitoxin significantly inhibited K2P3.1 and K2P16.1 channels. By contrast, digoxin displayed isolated inhibitory effects on K2P3.1. K2P3.1 outward currents were reduced by 80% (digitoxin, 1 Hz) and 78% (digoxin, 1 Hz). Digitoxin inhibited K2P3.1 currents with an IC50 value of 7.4 µM. Outward rectification properties of the channel were not affected. Mutagenesis studies revealed that amino acid residues located at the cytoplasmic site of the K2P3.1 channel pore form parts of a molecular binding site for cardiac glycosides. In conclusion, cardiac glycosides target human K2P channels. The antiarrhythmic significance of repolarizing atrial K2P3.1 current block by digoxin and digitoxin requires validation in translational and clinical studies.

Concepts: Amino acid, Atrial fibrillation, Action potential, Electrophysiology, Cardiac electrophysiology, Digoxin, Inward-rectifier potassium ion channel, Cardiac glycoside


Ibrutinib (IBT), the first-in-class inhibitor of Bruton’s tyrosine kinase (BTK), has demonstrated clinical activity against various B-cell malignancies. IBT is the second agent approved by U.S. Food and Drug Administration via Breakthrough Therapy Designation. Aside from its therapeutic mechanism through BTK inhibition, IBT has other target sites reported for cancer therapy, leading us to investigate whether IBT has unreported targets. Our study revealed that IBT can inhibit the growth of SMMC-7721 cells through irreversible inhibition of mammalian thioredoxin reductase (TrxR) enzymes, which are components of the thioredoxin (Trx) system. Further study demonstrated that IBT can cause cellular ROS elevation and induce oxidative stress-mediated apoptosis. The discovery of a new target of IBT sheds light on better understanding its anticancer mechanisms and provides a theoretical foundation for its further use in clinical therapy.