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1.  Lactate’s Effect on Human Neuroblastoma Cell Bioenergetic Fluxes 
Biochemical pharmacology  2015;99:88-100.
Lactate, once considered a metabolic dead-end, has been recently proposed to support neuron bioenergetics. To better understand how lactate specifically influences cell energy metabolism, we studied the effects of lactate supplementation on SH-SY5Y human neuroblastoma cell bioenergetic fluxes. Lactate supplementation increased cell respiration, there was no change in respiratory coupling efficiency, and lactate itself appeared to directly support the respiratory flux increase. Conversely, lactate supplementation reduced the glycolysis flux. This apparent pro-aerobic shift in the respiration:glycolysis ratio was accompanied by post-translational modifications and compartmental redistributions of proteins that respond to and modify bioenergetic fluxes, including cAMP-response element binding protein (CREB), p38 mitogen-activated protein kinases (p38 MAPK), AMP-activated protein kinase (AMPK), peroxisome-proliferator activated receptor gamma coactivator 1 β (PGC-1β), Akt, mammalian target of rapamycin (mTOR), and forkhead box protein O1 (FOXO1). mRNA levels for PGC-1β, nuclear respiratory factor 1 (NRF1), and cytochrome c oxidase subunit 1 (COX1) increased. Some effects depended on the direct presence of lactate, while others were durable and evident several hours after lactate was removed. We conclude lactate can be used to manipulate cell bioenergetics.
Graphical abstract
PMCID: PMC4706500  PMID: 26592660
Lactate; SH-SY5Y; mitochondrial respiration; glycolysis; bioenergetics
2.  In vitro and in vivo metabolism of N-adamantyl substituted urea-based soluble epoxide hydrolase inhibitors 
Biochemical pharmacology  2015;98(4):718-731.
N,N′-Disubstituted urea-based soluble epoxide hydrolase (sEH) inhibitors are promising therapeutics for hypertension, inflammation, and pain in multiple animal models. The drug absorption and pharmacological efficacy of these inhibitors have been reported extensively. However, the drug metabolism of these inhibitors is not well described. Here we reported the metabolic profile and associated biochemical studies of an N-adamantyl urea-based sEH inhibitor 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)pentyl)urea (AEPU) in vitro and in vivo. The metabolites of AEPU were identified by interpretation of liquid chromatography-mass chromatography (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or NMR. In vitro, AEPU had three major positions for phase I metabolism including oxidations on the adamantyl moiety, urea nitrogen atoms, and cleavage of the polyethylene glycol chain. In a rodent model, the metabolites from the hydroxylation on the adamantyl group and nitrogen atom were existed in blood while the metabolites from cleavage of polyethylene glycol chain were not found in urine. The major metabolite found in rodent urine was 3-(3-adamantyl-ureido)-propanoic acid, a presumably from cleavage and oxidation of the polyethylene glycol moiety. All the metabolites found were active but less potent than AEPU at inhibiting human sEH. Furthermore, cytochrome P450 (CYP) 3A4 was found to be a major enzyme mediating AEPU metabolism. In conclusion, the metabolism of AEPU resulted from oxidation by CYP could be shared with other N-adamantyl-urea-based compounds. These findings suggest possible therapeutic roles for AEPU and new strategies for drug design in this series of possible drugs.
Graphical Abstract
PMCID: PMC4656071  PMID: 26494425
soluble epoxide hydrolase; drug metabolism; LC-MS/MS; NMR; cytochrome P450s
3.  NOSH-aspirin (NBS-1120), a novel nitric oxide- and hydrogen sulfide-releasing hybrid has enhanced chemo-preventive properties compared to aspirin, is gastrointestinal safe with all the classic therapeutic indications 
Biochemical pharmacology  2015;98(4):564-572.
Aspirin is chemopreventive; however, side effects preclude its long-term use. NOSH-aspirin (NBS-1120), a novel hybrid that releases nitric oxide and hydrogen sulfide, was designed to be a safer alternative. Here we compare the gastrointestinal safety, anti-inflammatory, analgesic, antipyretic, anti-platelet, and chemopreventive properties of aspirin and NBS-1120 administered orally to rats at equimolar doses. Gastrointestinal safety: 6h post-administration, the number and size of hemorrhagic lesions in stomachs were counted; tissue samples were frozen for PGE2, SOD, and MDA determination. Anti-inflammatory: 1h after drug administration, the volume of carrageenan-induced rat paw edemas was measured for 5h. Anti-pyretic: fever was induced by LPS (ip) an hour before administration of the test drugs, core body temperature was measured hourly for 5h. Analgesic: time-dependent analgesic effects were evaluated by carrageenan-induced hyperalgesia. Antiplatelet: anti-aggregatory effects were studied on collagen-induced platelet aggregation of human platelet-rich plasma. Chemoprevention: Nude mice were gavaged daily for 25 days with vehicle, aspirin or NBS-1120. After one week, each mouse was inoculated subcutaneously in the right flank with HT-29 human colon cancer cells. Both agents reduced PGE2 levels in stomach tissue; however, NBS-1120 did not cause any stomach ulcers, whereas aspirin caused significant bleeding. Lipid peroxidation induced by aspirin was higher than that exerted by NBS-1120. SOD activity was significantly inhibited by aspirin but increased by NBS-1120. Both agents showed similar anti-inflammatory, analgesic, anti-pyretic, and anti-platelet activities. Aspirin increased plasma TNFα more than NBS-1120-treated animals. NBS-1120 was better than aspirin as a chemopreventive agent; it dose-dependently inhibited tumor growth and tumor mass.
PMCID: PMC4656078  PMID: 26394025
Nitric oxide; hydrogen sulfide; aspirin; GI-sparing; chemopreventive
4.  Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling 
Biochemical pharmacology  2015;98(4):614-628.
Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40 mM K+, or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.
PMCID: PMC4656099  PMID: 26549367
NSAID; Cell migration; Membrane potential; Voltage-gated potassium channel; Calpain
5.  Radiometric Assay of Ghrelin Hydrolase Activity and 3H-Ghrelin Distribution into Mouse Tissues 
Biochemical pharmacology  2015;98(4):732-739.
A high-throughput radiometric assay was developed to characterize enzymatic hydrolysis of ghrelin and to track the peptide's fate in vivo. The assay is based on solvent partitioning of [3H]-octanoic acid liberated from [3H]-octanoyl ghrelin during enzymatic hydrolysis. This simple and cost-effective method facilitates kinetic analysis of ghrelin hydrolase activity of native and mutated butyrylcholinesterases or carboxylesterases from multiple species. In addition, the assay's high sensitivity facilitates ready evaluation of ghrelin's pharmacokinetics and tissue distribution in mice after i.v. bolus administration of radiolabeled peptide.
Graphical Abstract
PMCID: PMC4669970  PMID: 26514871
Butyrylcholinesterase; Ghrelin; Radiometric assay; Enzyme kinetics; Pharmacokinetics
6.  Combination therapy with bioengineered miR-34a prodrug and doxorubicin synergistically suppresses osteosarcoma growth 
Biochemical pharmacology  2015;98(4):602-613.
Osteosarcoma (OS) is the most common form of primary malignant bone tumor and prevalent among children and young adults. Recently we have established a novel approach to bioengineering large quantity of microRNA-34a (miR-34a) prodrug for miRNA replacement therapy. This study is to evaluate combination treatment with miR-34a prodrug and doxorubicin, which may synergistically suppress human OS cell growth via RNA interference and DNA intercalation. Synergistic effects were indeed obvious between miR-34a prodrug and doxorubicin for the suppression of OS cell proliferation, as defined by Chou-Talalay method. The strongest antiproliferative synergism was achieved when both agents were administered simultaneously to the cells at early stage, which was associated with much greater degrees of late apoptosis, necrosis, and G2 cell cycle arrest. Alteration of OS cellular processes and invasion capacity was linked to the reduction of protein levels of miR-34a targeted (proto-)oncogenes including SIRT1, c-MET, and CDK6. Moreover, orthotopic OS xenograft tumor growth was repressed to a significantly greater degree in mouse models when miR-34a prodrug and doxorubicin were co-administered intravenously. In addition, multiple doses of miR-34a prodrug and doxorubicin had no or minimal effects on mouse blood chemistry profiles. The results demonstrate that combination of doxorubicin chemotherapy and miR-34a replacement therapy produces synergistic antiproliferative effects and it is more effective than monotherapy in suppressing OS xenograft tumor growth. These findings support the development of mechanism-based combination therapy to combat OS and bioengineered miR-34a prodrug represents a new natural miRNA agent.
PMCID: PMC4725324  PMID: 26518752
Osteosarcoma; miR-34a; doxorubicin; chemotherapy; orthotopic tumor
7.  Functional Expression of the 11 Human Organic Anion Transporting Polypeptides in Insect Cells Reveals that Sodium Fluorescein is a General OATP Substrate 
Biochemical pharmacology  2015;98(4):649-658.
Organic Anion Transporting Polypeptides (OATPs), encoded by genes of the Solute Carrier Organic Anion (SLCO) family, are transmembrane proteins involved in the uptake of various compounds of endogenous or exogenous origin. In addition to their physiological roles, OATPs influence the pharmacokinetics and drug-drug interactions of several clinically relevant compounds. To examine the function and molecular interactions of human OATPs, including several poorly characterized family members, we expressed all 11 human OATPs at high levels in the baculovirus-Sf9 cell system. We measured the temperature- and inhibitor-sensitive cellular accumulation of sodium fluorescein and fluorescein-methotrexate, two fluorescent substrates of the OATPs, OATP1B1 and 1B3. OATP1B1 and 1B3 were functional in Sf9 cells, showing rapid uptake (t1/2(fluorescein-methotrexate) 2.64 and 4.16 min, and t1/2(fluorescein) 6.71 and 5.58 min for OATP1B1 and 1B3, respectively) and high-affinity transport (Km(fluorescein-methotrexate) 0.23 and 0.53 μM, and Km(fluorescein) 25.73 and 38.55 μM for OATP1B1 and 1B3, respectively) of both substrates. We found that sodium fluorescein is a general substrate of all human OATPs: 1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, while fluorescein-methotrexate is only transported by 1B1, 1B3, 1A2 and 2B1. Acidic extracellular pH greatly facilitated fluorescein uptake by all OATPs, and new molecular interactions were detected (between OATP2B1 and Imatinib, OATP3A1, 5A1 and 6A1 and estradiol 17-β–D-glucuronide, and OATP1C1 and 4C1 and prostaglandin E2). These studies demonstrate for the first time the applicability of the insect cell system for the functional expression of the entire human OATP family, and for drug-OATP interaction screening.
PMCID: PMC4959548  PMID: 26415544
Organic Anion Transporting Polypeptide; fluorescent assay; new substrates; drug screening
8.  New paradigms in GPCR drug discovery 
Biochemical pharmacology  2015;98(4):541-555.
G protein-coupled receptors (GPCRs) remain a major domain of pharmaceutical discovery. The discovery of GPCR lead compounds and their optimization are now structure-based, thanks to advances in X-ray crystallography, molecular modeling, protein engineering and biophysical techniques. In silico screening provides useful hit molecules. New pharmacological approaches to tuning the pleotropic action of GPCRs include: allosteric modulators, biased ligands, GPCR heterodimer-targeted compounds, manipulation of polypharmacology, receptor antibodies and tailoring of drug molecules to fit GPCR pharmacogenomics. Measurements of kinetics and drug efficacy are factors influencing clinical success. With the exception of inhibitors of GPCR kinases, targeting of intracellular GPCR signaling or receptor cycling for therapeutic purposes remains a futuristic concept. New assay approaches are more efficient and multidimensional: cell-based, label-free, fluorescence-based assays, and biosensors. Tailoring GPCR drugs to a patient’s genetic background is now being considered. Chemoinformatic tools can predict ADME-tox properties. New imaging technology visualizes drug action in vivo. Thus, there is reason to be optimistic that new technology for GPCR ligand discovery will help improve the current narrowing of the pharmaceutical pipeline.
PMCID: PMC4967540  PMID: 26265138
Drug discovery; GPCR; X-ray crystallography; structure-based design; signaling; inhibitors
9.  A metabolomic perspective of griseofulvin-induced liver injury in mice 
Biochemical pharmacology  2015;98(3):493-501.
Griseofulvin (GSF) causes hepatic porphyria in mice, which mimics the liver injury associated with erythropoietic protoporphyria (EPP) in humans. The current study investigated the biochemical basis of GSF-induced liver injury in mice using a metabolimic approach. GSF treatment in mice resulted in significant accumulations of protoporphyrin IX (PPIX), N-methyl PPIX, bile acids, and glutathione (GSH) in the liver. Metabolomic analysis also revealed bioactivation pathways of GSF that contributed to the formation of GSF-PPIX, GSF-GSH and GSF-proline adducts. GSF-PPIX is the precursor of N-methyl PPIX. A six-fold increase of N-methyl PPIX was observed in the liver of mice after GSF treatment. N-methyl PPIX strongly inhibits ferrochelatase, the enzyme that converts PPIX to heme, and leads to PPIX accumulation. Excessive PPIX in the liver results in bile duct blockage and disturbs bile acid homeostasis. The accumulation of GSH in the liver was likely due to Nrf2-mediated upregulation of GSH synthesis. In summary, this study provides the biochemical basis of GSF-induced liver injury that can be used to understand the pathophysiology of EPP-associated liver injury in humans.
Graphical Abstract
PMCID: PMC4699580  PMID: 26343413
Griseofulvin; drug toxicity; drug metabolism; metabolomics; erythropoietic protoporphyria
10.  Copper and protons directly activate the zinc-activated channel 
Biochemical pharmacology  2016;103:109-117.
The zinc-activated channel (ZAC) is a cationic ion channel belonging to the superfamily of Cys-loop receptors, which consists of pentameric ligand-gated ion channels. ZAC is the least understood member of this family so in the present study we sought to characterize the properties of this channel further. We demonstrate that not only zinc (Zn2+) but also copper (Cu2+) and protons (H+) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H+ > Cu2+ > Zn2+ and H+ > Zn2+ > Cu2+, respectively. The responses elicited by Zn2+, Cu2+ and H+ through ZAC are all characterized by low degrees of desensitization. In contrast, currents evoked by high concentrations of the three agonists comprise distinctly different activation and decay components, with transitions to and from an open state being significantly faster for H+ than for the two metal ions. The permeabilities of ZAC for Na+ and K+ relative to Cs+ are indistinguishable, whereas replacing all of extracellular Na+ and K+ with the divalent cations Ca2+ or Mg2+ results in complete elimination of Zn2+-activated currents at both negative and positive holding potentials. This indicates that ZAC is non-selectively permeable to monovalent cations, whereas Ca2+ and Mg2+ inhibit the channel. In conclusion, this is the first report of a Cys-loop receptor being gated by Zn2+, Cu2+ and H+. ZAC could be an important mediator of some of the wide range of physiological functions regulated by or involving Zn2+, Cu2+ and H+.
PMCID: PMC5119521  PMID: 26872532
Zinc; Copper; Protons; Cys-loop ligand-gated ion channels; Calcium block
11.  Genome-wide Analysis of Human Constitutive Androstane Receptor (CAR) Transcriptome in Wild-type and CAR-knockout HepaRG cells 
Biochemical pharmacology  2015;98(1):190-202.
The constitutive androstane receptor (CAR) modulates the transcription of numerous genes involving drug metabolism, energy homeostasis, and cell proliferation. Most functions of CAR however were defined from animal studies. Given the known species difference of CAR and the significant cross-talk between CAR and the pregnane X receptor (PXR), it is extremely difficult to decipher the exact role of human CAR (hCAR) in gene regulation, relying predominantly on pharmacological manipulations. Here, utilizing a newly generated hCAR-knockout (KO) HepaRG cell line, we carried out RNA-seq analysis of the global transcriptomes in wild-type (WT) and hCAR-KO HepaRG cells treated with CITCO, a selective hCAR agonist, phenobarbital (PB), a dual activator of hCAR and hPXR, or vehicle control. Real-time PCR assays in separate experiments were used to validate RNA-seq findings. Our results indicate that genes encoding drug-metabolizing enzymes are among the main clusters altered by both CITCO and PB. Specifically, CITCO significantly changed the expression of 135 genes in an hCAR-dependent manner, while PB altered the expression of 227 genes in WT cells of which 94 were simultaneously modulated in both cell lines reflecting dual effects of PB on hCAR/PXR. Notably, we found that many genes promoting cell proliferation and tumorigenesis were up-regulated in hCAR-KO cells, suggesting that hCAR may play an important role in cell growth that differs from mouse CAR. Together, our results reveal both novel and known targets of hCAR and support the role of hCAR in maintaining the homeostasis of metabolism and cell proliferation in the liver.
PMCID: PMC4600658  PMID: 26275810
HepaRG; CAR; gene knockout; RNA-seq; phenobarbital; CITCO
12.  Toll-like receptor 4 signaling: A common pathway for interactions between prooxidants and extracellular disulfide high mobility group box 1 (HMGB1) protein-coupled activation 
Biochemical pharmacology  2015;98(1):132-143.
Necrotic cells passively release HMGB1, which can stimulate TLR4 in an autocrine fashion to potentially initiate “sterile” inflammation that maintains different disease states. We have shown that prooxidants can induce NF-κB activation through TLR4 stimulation. We examined whether prooxidants enhance HMGB1-induced TLR4 signaling through NF-κB activation. We used LPS-EK as a specific agonist for TLR4, and PPC and SIN-1 as in situ sources for ROS. As model systems, we used HEK-Blue cells (stably transfected with mouse TLR4), RAW-Blue™ cells (derived from murine RAW 264.7 macrophages) and primary murine macrophages from TLR4-KO mice. Both HEK-Blue and RAW-Blue 264.7 cells express optimized secreted embryonic alkaline phosphatase (SEAP) reporter under the control of a promoter inducible by NF-κB. We treated cells with HMGB1 alone and/or in conjunction with prooxidants and/or inhibitors using SEAP release as a measure of TLR4 stimulation. HMGB1 alone and/or in conjunction with prooxidants increased TNFα and IL-6 released from TLR4-WT, but not from TLR4-KO macrophages. Pro-oxidants increased HMGB1 release, which we quantified by ELISA. We used both fluorescence microscopy imaging and flow cytometry to quantify the expression of intracellular ROS. TLR4-neutralizing antibody decreased prooxidant-induced HMGB1 release. Prooxidants promoted HMGB1-induced NF-κB activation as determined by increased release of SEAP and TNF-α, and accumulation of iROS. HMGB1 (Box A), anti-HMGB1 and anti-TLR4-neutralizing pAbs inhibited HMGB1-induced NF-κB activation, but HMGB1 (Box A) and anti-HMGB1 pAb had no effect on prooxidant-induced SEAP release. The present results confirm that prooxidants enhance proinflammatory effects of HMGB1 by activating NF-κB through TLR4 signaling.
PMCID: PMC4600681  PMID: 26367307
Toll-like receptor 4; Prooxidants; Recombinant high mobility group box 1 protein; NF-κB activation; sterile inflammation
13.  Chemical inhibition of fatty acid absorption and cellular uptake limits lipotoxic cell death 
Biochemical pharmacology  2015;98(1):167-181.
Chronic elevation of plasma free fatty acid (FFA) levels is commonly associated with obesity, type 2 diabetes, cardiovascular disease and some cancers. Experimental evidence indicates FFA and their metabolites contribute to disease development through lipotoxicity. Previously, we identified a specific fatty acid transport inhibitor CB16.2, a.k.a. Lipofermata, using high throughput screening methods. In this study, efficacy of transport inhibition was measured in four cell lines that are models for myocytes (mmC2C12), pancreatic ß-cells (rnINS-1E), intestinal epithelial cells (hsCaco-2), and hepatocytes (hsHepG2), as well as primary human adipocytes. The compound was effective in inhibiting uptake with IC50s between 3 and 6 µM for all cell lines except human adipocytes (39 µM). Inhibition was specific for long and very long chain fatty acids but had no effect on medium chain fatty acids (C6-C10), which are transported by passive diffusion. Derivatives of Lipofermata were evaluated to understand structural contributions to activity. Lipofermata prevented palmitate-mediated oxidative stress, induction of BiP and CHOP, and cell death in a dose-dependent manner in hsHepG2 and rnINS-1E cells, suggesting it will prevent induction of fatty acid-mediated cell death pathways and lipotoxic disease by channeling excess fatty acids to adipose tissue and away from liver and pancreas. Importantly, mice dosed orally with Lipofermata were not able to absorb 13C-oleate demonstrating utility as an inhibitor of fatty acid absorption from the gut.
PMCID: PMC4610366  PMID: 26394026
FATP2 Inhibitor; Lipotoxicity; Lipid Droplet; Fatty acid transport
14.  Adipose extracellular matrix remodelling in obesity and insulin resistance☆ 
Biochemical pharmacology  2016;119:8-16.
The extracellular matrix (ECM) of adipose tissues undergoes constant remodelling to allow adipocytes and their precursor cells to change cell shape and function in adaptation to nutritional cues. Abnormal accumulation of ECM components and their modifiers in adipose tissues has been recently demonstrated to cause obesity-associated insulin resistance, a hallmark of type 2 diabetes. Integrins and other ECM receptors (e.g. CD44) that are expressed in adipose tissues have been shown to regulate insulin sensitivity. It is well understood that a hypoxic response is observed in adipose tissue expansion during obesity progression and that hypoxic response accelerates fibrosis and inflammation in white adipose tissues. The expansion of adipose tissues should require angiogenesis; however, the excess deposition of ECM limits the angiogenic response of white adipose tissues in obesity. While recent studies have focused on the metabolic consequences and the mechanisms of adipose tissue expansion and remodelling, little attention has been paid to the role played by the interaction between peri-adipocyte ECM and their cognate cell surface receptors. This review will address what is currently known about the roles played by adipose ECM, their modifiers, and ECM receptors in obesity and insulin resistance. Understanding how excess ECM deposition in the adipose tissue deteriorates insulin sensitivity would provide us hints to develop a new therapeutic strategy for the treatment of insulin resistance and type 2 diabetes.
PMCID: PMC5061598  PMID: 27179976
Adipose tissue; Extracellular matrix; Integrins; Insulin resistance; Fibrosis
15.  Presence of multiple binding sites on α9α10 nAChR receptors alludes to stoichiometric dependent action of the α-conotoxin, Vc1.1 
Biochemical pharmacology  2014;89(1):131-140.
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in fast synaptic transmission. nAChRs are pentameric receptors formed from a combination of different or similar subunits to produce heteromeric or homomeric channels. The heteromeric, α9α10 nAChR subtype is well-known for its role in the auditory system, being expressed in cochlear hair cells. These nAChRs have also been shown to be involved in immune-modulation. Antagonists of α9α10 nAChRs, like the α-conotoxin Vc1.1, have analgesic effects in neuropathic pain. Unlike other nAChR subtypes there is no evidence that functional receptor stoichiometries of α9α10 exist. By using 2-electrode voltage clamp methods and maintaining a constant intracellular Ca2+ concentration, we observed a biphasic activation curve for ACh that is dependent on receptor stoichiometry. Vc1.1, but not the α9α10 antagonists RgIA or atropine, inhibits ACh-evoked currents in a biphasic manner. Characteristics of the ACh and Vc1.1 activation and inhibition curves can be altered by varying the ratio of α9 and α10 mRNA injected into oocytes, changing the curves from biphasic to monophasic when an excess of α10 mRNA is used. These results highlight the difference in the pharmacological profiles of at least two different α9α10 nAChR stoichiometries, possibly (α9)3(α10)2 and (α9)2(α10)3. As a result, we infer that there is an additional binding site for ACh and Vc1.1 at the α9-α9 interface on the hypothesized (α9)3(α10)2 nAChR, in addition to the α10-α9 and or α9-α10 interfaces that are common to both stoichiometries. This study provides further evidence that receptor stoichiometry contributes another layer of complexity in understanding Cys-loop receptors.
Graphical Abstract
PMCID: PMC5089836  PMID: 24548457
Nicotinic acetylcholine receptors; subunit stoichiometry; α-conotoxins; Vc1.1; RgIA; alpha9alpha10
16.  Modulation of aggressive behavior in mice by nicotinic receptor subtypes 
Biochemical pharmacology  2015;97(4):488-497.
Aggression is frequently comorbid with neuropsychiatric conditions and is a predictor of worse outcomes, yet current pharmacotherapies are insufficient and have debilitating side effects, precluding broad use. Multiple models of aggression across species suggest that the nicotinic acetylcholine receptor (nAChR) agonist nicotine has anti-aggressive (serenic) properties. Here we demonstrate dose-dependent serenic effects of acute nicotine administration in three distinct mouse strains: C57BL/6, BALB/c, and CD1. While acute nicotine administration (0.25 mg/kg) modestly reduced solitary homecage locomotion, this could not account for nicotine’s serenic effects since social encounters eliminated the hypolocomotor effect, and nicotine did not alter social interaction times. Pretreatment with the homomeric (α7 subunit) nAChR antagonist methyllycaconitine (5 mg/kg), but not the heteromeric (β2 or β4 subunit-containing) nAChR antagonist dihydro-β-erythroidine (DHβE, 3 mg/kg), blocked the serenic effects of nicotine. By contrast, pretreatment with DHβE blocked the effect of acute nicotine administration on locomotion, uncoupling nicotine’s serenic and hypolocomotor effects. Finally, the α7 nAChR partial agonist GTS-21 reduced aggression in C57BL/6 mice. These results support the idea that acute nicotine administration has serenic effects and provide evidence for specificity of this effect distinct from effects on locomotion. Furthermore, pharmacological studies suggest that activation of α7 nAChRs underlies the serenic effects of nicotine. Further studies of nAChRs could enhance understanding of the neurobiology of aggression and may lead to the development of novel, more specific treatments for pathological aggression.
Graphical abstract
PMCID: PMC4600457  PMID: 26212554
aggression; nicotinic acetylcholine receptor; nicotine; GTS-21; DMXB; α7
17.  The role of alpha5 nicotinic acetylcholine receptors in mouse models of chronic inflammatory and neuropathic pain 
Biochemical pharmacology  2015;97(4):590-600.
The aim of the present study was to determine the impact of α5 nicotinic acetylcholine receptor (nAChR) subunit deletion in the mouse on the development and intensity of nociceptive behavior in various chronic pain models.
The role of α5-containing nAChRs was explored in mouse models of chronic pain, including peripheral neuropathy (chronic constriction nerve injury, CCI), tonic inflammatory pain (the formalin test) and short and long-term inflammatory pain (complete Freund’s adjuvant, CFA and carrageenan tests) in α5 knock-out (KO) and wild-type (WT) mice.
The results showed that paw-licking time was decreased in the formalin test, and the hyperalgesic and allodynic responses to carrageenan and CFA injections were also reduced. In addition, paw edema in formalin-, carrageenan- or CFA-treated mice were attenuated in α5-KO mice significantly. Furthermore, tumor necrosis factor-alpha (TNF-α) levels of carrageenan-treated paws were lower in α5-KO mice. The antinociceptive effects of nicotine and sazetidine-A but not varenicline were α5-dependent in the formalin test. Both hyperalgesia and allodynia observed in the CCI test were reduced in α5-KO mice. Nicotine reversal of mechanical allodynia in the CCI test was mediated through α5-nAChRs at spinal and peripheral sites.
In summary, our results highlight the involvement of the α5 nAChR subunit in the development of hyperalgesia, allodynia and inflammation associated with chronic neuropathic and inflammatory pain models. They also suggest the importance of α5-nAChRs as a target for the treatment of chronic pain.
Graphical Abstract
PMCID: PMC4600420  PMID: 25931144
alpha 5; nicotinic acetylcholine receptors; neuropathic pain; inflammatory pain
18.  The Human Clinical Phenotypes of Altered CHRNA7 Copy Number 
Biochemical pharmacology  2015;97(4):352-362.
Copy number variants (CNVs) have been implicated in multiple neuropsychiatric conditions, including autism spectrum disorder (ASD), schizophrenia, and intellectual disability (ID). Chromosome 15q13 is a hotspot for such CNVs due to the presence of low copy repeat (LCR) elements, which facilitate non-allelic homologous recombination (NAHR). Several of these CNVs have been overrepresented in individuals with neuropsychiatric disorders; yet variable expressivity and incomplete penetrance are commonly seen. Dosage sensitivity of the CHRNA7 gene, which encodes for the α7 nicotinic acetylcholine receptor in the human brain, has been proposed to have a major contribution to the observed cognitive and behavioral phenotypes, as it represents the smallest region of overlap to all the 15q13.3 deletions and duplications. Individuals with zero to four copies of CHRNA7 have been reported in the literature, and represent a range of clinical severity, with deletions causing generally more severe and more highly penetrant phenotypes. Potential mechanisms to account for the variable expressivity within each group of 15q13.3 CNVs will be discussed.
Graphical abstract
PMCID: PMC4600432  PMID: 26095975
Nicotinic acetylcholine receptors; CHRNA7; copy number variation; neuropsychiatric disease
19.  Long-lasting Changes in Neural Networks to Compensate for Altered Nicotinic Input 
Biochemical pharmacology  2015;97(4):418-424.
The nervous system must balance excitatory and inhibitory input to constrain network activity levels within a proper dynamic range. This is a demanding requirement during development when networks form and throughout adulthood as networks respond to constantly changing environments. Defects in the ability to sustain a proper balance of excitatory and inhibitory activity are characteristic of numerous neurological disorders such as schizophrenia, Alzheimer’s disease, and autism. A variety of homeostatic mechanisms appear to be critical for balancing excitatory and inhibitory activity in a network. These are operative at the level of individual neurons, regulating their excitability by adjusting the numbers and types of ion channels, and at the level of synaptic connections, determining the relative numbers of excitatory versus inhibitory connections a neuron receives. Nicotinic cholinergic signaling is well positioned to contribute at both levels because it appears early in development, extends across much of the nervous system, and modulates transmission at many kinds of synapses. Further, it is known to influence the ratio of excitatory-to-inhibitory synapses formed on neurons during development. GABAergic inhibitory neurons are likely to be key for maintaining network homeostasis (limiting excitatory output), and nicotinic signaling is known to prominently regulate the activity of several GABAergic neuronal subtypes. But how nicotinic signaling achieves this and how networks may compensate for the loss of such input are important questions remaining unanswered. These issues are reviewed.
Graphical Abstract
PMCID: PMC4600434  PMID: 26206188
Nicotinic; homeostasis; compensation; neural network; E/I ratio; circuits
20.  Activation of endplate nicotinic acetylcholine receptors by agonists 
Biochemical pharmacology  2015;97(4):601-608.
The interaction of a small molecule made in one cell with a large receptor made in another is the signature event of cell signaling. Understanding the structure and energy changes associated with agonist activation is important for engineering drugs, receptors and synapses. The nicotinic acetylcholine receptor (AChR) is a ~300 kD ion channel that binds the neurotransmitter acetylcholine (ACh) and other cholinergic agonists to elicit electrical responses in the central and peripheral nervous systems. This mini-review is in two sections. First, general concepts of skeletal muscle AChR operation are discussed in terms of energy landscapes for conformational change. Second, adult vs. fetal AChRs are compared with regard to interaction energies between ACh and agonist-site side chains, measured by single-channel electrophysiology and molecular dynamics simulations. The five aromatic residues that form the core of each agonist binding site can be divided into two working groups, a triad (led by αY190) that behaves similarly at all sites and a coupled pair (led by γW55) that has a large influence on affinity only in fetal AChRs.
Each endplate AChR has 5 homologous subunits, two of α(1) and one each of β, δ and either γ (fetal) or ε (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at αδ and either αγ or αε subunit interfaces. The receptor undergoes a reversible, global isomerization between structures called C and O. The C shape does not conduct ions and has a relatively low affinity for ACh, whereas O conducts cations and has a higher affinity. When both agonist sites are empty (filled only with water) the probability of taking on the O conformation (PO) is low, <10−6. When ACh molecules occupy the agonist sites the C→O opening rate constant and C↔O gating equilibrium constant increase dramatically. Following a pulse of ACh at the nerve-muscle synapse, the endplate current rises rapidly to reach a peak that corresponds to PO ~0.96.
PMCID: PMC4600445  PMID: 26206191
nicotinic; acetylcholine; neuromuscular; allosteric; gating; binding
21.  The effect of α7 nicotinic receptor activation on glutamatergic transmission in the hippocampus 
Biochemical pharmacology  2015;97(4):439-444.
Nicotinic acetylcholine receptors (nAChRs) are expressed widely in the CNS, and mediate both synaptic and perisynaptic activities of endogenous cholinergic inputs and pharmacological actions of exogenous compounds (e.g. nicotine and choline). Behavioral studies indicate that nicotine improves such cognitive functions as learning and memory, however the cellular mechanism of these actions remains elusive. With help from newly developed biosensors and optogenetic tools, recent studies provide new insights on signaling mechanisms involved in the activation of nAChRs. Here we will review α7 nAChR’s action in the tri-synaptic pathway in the hippocampus. The effects of α7 nAChR activation via either exogenous compounds or endogenous cholinergic innervation are detailed for spontaneous and evoked glutamatergic synaptic transmission and synaptic plasticity, as well as the underlying signaling mechanisms. In summary, α7 nAChRs trigger intracellular calcium rise and calcium-dependent signaling pathways to enhance glutamate release and induce glutamatergic synaptic plasticity.
Graphical abstract
PMCID: PMC4600449  PMID: 26212541
22.  Alpha7 nicotinic receptors as therapeutic targets for Parkinson’s disease 
Biochemical pharmacology  2015;97(4):399-407.
Accumulating evidence suggests that CNS α7 nicotinic acetylcholine receptors (nAChRs) are important targets for the development of therapeutic approaches for Parkinson’s disease. This progressive neurodegenerative disorder is characterized by debilitating motor deficits, as well as autonomic problems, cognitive declines, changes in affect and sleep disturbances. Currently L-dopa is the gold standard treatment for Parkinson’s disease motor problems, particularly in the early disease stages. However, it does not improve the other symptoms, nor does it reduce the inevitable disease progression. Novel therapeutic strategies for Parkinson’s disease are therefore critical. Extensive pre-clinical work using a wide variety of experimental models shows that nicotine and nAChR agonists protect against damage to nigrostriatal and other neuronal cells. This observation suggests that nicotine and/or nAChR agonists may be useful as disease modifying agents. Additionally, studies in several parkinsonian animal models including nonhuman primates show that nicotine reduces L-dopa-induced dyskinesias, a side effect of L-dopa therapy that may be as incapacitating as Parkinson’s disease itself. Work with subtype selective nAChR agonists indicate that α7 nAChRs are involved in mediating both the neuroprotective and antidyskinetic effects, thus offering a targeted strategy with optimal beneficial effects and minimal adverse responses. Here, we review studies demonstrating a role for α7 nAChRs in protection against neurodegenerative effects and for the reduction of L-dopa-induced dyskinesias. Altogether, this work suggests that α7 nAChRs may be useful targets for reducing Parkinson’s disease progression and for the management of the dyskinesias that arise with L-dopa therapy.
Graphical Abstract
PMCID: PMC4600450  PMID: 26093062
Alpha7; L-dopa-induced dyskinesias; Neuroprotection; Nicotinic receptors; Parkinson’s disease
23.  Nicotine Modulation of Fear Memories and Anxiety: Implications for Learning and Anxiety Disorders 
Biochemical pharmacology  2015;97(4):498-511.
Anxiety disorders are a group of crippling mental diseases affecting millions of Americans with a 30% lifetime prevalence and costs associated with healthcare of $42.3 billion. While anxiety disorders show high levels of co-morbidity with smoking (45.3% vs. 22.5% in healthy individuals), anxiety disorders are also more common among the smoking population (22% vs. 11.1% in the non-smoking population). Moreover, there is clear evidence that smoking modulates symptom severity in patients with anxiety disorders. In order to better understand this relationship, several animal paradigms are used to model several key symptoms of anxiety disorders; these include fear conditioning and measures of anxiety. Studies clearly demonstrate that nicotine mediates acquisition and extinction of fear as well as anxiety through the modulation of specific subtypes of nicotinic acetylcholine receptors (nAChRs) in brain regions involved in emotion processing such as the hippocampus. However, the direction of nicotine’s effects on these behaviors is determined by several factors that include the length of administration, hippocampus-dependency of the fear learning task, and source of anxiety (novelty-driven vs. social anxiety). Overall, the studies reviewed here suggest that nicotine alters behaviors related to fear and anxiety and that nicotine contributes to the development, maintenance, and reoccurrence of anxiety disorders.
PMCID: PMC4600451  PMID: 26231942
Nicotine; Anxiety Disorders; Fear Conditioning; Anxiety; Extinction
24.  Development of [18F]ASEM, a specific radiotracer for quantification of the α7-nAChR with positron-emission tomography 
Biochemical pharmacology  2015;97(4):566-575.
The alpha-7 subtype of the nicotinic acetylcholine receptor (α7-nAChR) is fundamental to physiology; it mediates various brain functions and represents an important target for drug discovery. Exploration of the brain nicotinic acetylcholine receptors (nAChRs) using positron-emission tomography (PET) will make it possible to better understand the important role of this receptor and to study its involvement in schizophrenia, bipolar disorder, Alzheimer's and Parkinson's diseases, drug dependence, inflammation and many other disorders and simplify the development of nicotinic drugs for treatment of these disorders.
Until recently, PET imaging of α7-nAChRs has been impeded by the absence of good radiotracers. This review describes various endeavors to develop α7-nAChR PET tracers by several research groups including the author’s group. Most initial PET tracers for imaging α7-nAChRs did not exhibit suitable imaging properties due to their low specific binding. Recently discovered [18F]ASEM is the first highly specific α7-nAChR radioligand and it was recently translated to human PET imaging.
PMCID: PMC4600455  PMID: 26232729
positron emission tomography; PET; α7-nAChR; alpha 7; nicotinic receptor; [18F]ASEM
25.  High Affinity α3β4 Nicotinic Acetylcholine Receptor Ligands AT-1001 and AT-1012 Attenuate Cocaine-Induced Conditioned Place Preference and Behavioral Sensitization in Mice 
Biochemical pharmacology  2015;97(4):531-541.
Cholinergic signaling via the nicotinic acetylcholine receptors (nAChRs) in the mesolimbic circuitry is involved in the rewarding effects of abused drugs such as cocaine and opioids. In mouse studies, nonselective nAChR antagonist mecamylamine blocks cocaine-induced conditioned place preference (CPP) and behavioral sensitization. Among subtype-selective nAChR antagonists, the β2-selective antagonist dihydrobetaerythroidine and α7 nAChR antagonist methyllycaconitine (MLA), but not MLA alone prevent behavioral sensitization to cocaine. Since the role of the α3β4 nAChR subtype in the rewarding and behavioral effects of cocaine is unknown, the present study investigated the effect of two potent and selective α3β4 nAChR ligands, AT-1001 and AT-1012, on the acquisition of cocaine-induced CPP and behavioral sensitization in mice. At 5–30 mg/kg, cocaine produced robust CPP, whereas behavioral sensitization of locomotor activity was only observed at the higher doses (20–30 mg/kg). Pretreatment with AT-1001 (1–10 mg/kg) or AT-1012 (3–10 mg/kg) blocked CPP induced by 5 mg/kg cocaine, but not by 30 mg/kg cocaine. Lower doses of AT-1001 (0.3–3 mg/kg) and AT-1012 (1–3 mg/kg) did not affect the increase in locomotor activity induced by 5 or 30 mg/kg cocaine. But AT-1001, at these doses, blocked locomotor sensitization induced by 30 mg/kg cocaine. These results indicate that the α3β4 nAChR play a role in the rewarding and behavioral effects of cocaine, and that selective α3β4 nAChR ligands can attenuate cocaine-induced behavioral phenomena. Since the selective α3β4 nAChR functional antagonist AT-1001 has also been shown to block nicotine self-administration in rats, the present results suggest that α3β4 nAChRs may be a target for the treatment of cocaine addiction as well as for cocaine-nicotine comorbid addiction.
Graphical abstract
PMCID: PMC4600456  PMID: 26256075
α3β4 nAChRs; cocaine-induced behavior; α3β4 nAChR-selective ligands; cocaine-induced conditioned place preference; cocaine addiction

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