The generation of rat dorsal root ganglion cell lines to identify the target of KW-7158, a novel treatment for overactive bladder

Nishiya, Yoichi; Yokokawa, Sachiko; Fukuda, Ayumu; Yamagata, Tsuyoshi; Inayoshi, Atsushi; Obinata, Masuo; Shibata, Kenji

doi:10.1016/j.neures.2011.07.1823

Cited by 2 publications

(10 citation statements)

References 27 publications

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“…Cell Culture TRD-10/13 cells were recloned by limiting dilution from TRD-10 cells 6) for the stable and maximal expression of the target molecule, as determined by a radio-ligand binding assay. The cells were cultured as described in our previous report.…”

Section: Methodsmentioning

confidence: 99%

“…The cells were cultured as described in our previous report. 6) cDNA Screening Each clone of the cDNA expression library 7) was separately transfected into TRD-49 cells with Lipofectamine 2000 (Invitrogen). After two days, the cells were washed, and binding buffer containing 1 µM of K-025-Cy3 and 0.0028% of digitonin was added.…”

Section: Methodsmentioning

confidence: 99%

“…6) Briefly, after rat bladder strips were equilibrated in Krebs-Henseleit solution, 1 µM of atropine and 3 µM of guanethidine were added to the organ bath. Then, the stimulation cycles were performed four times as follows: three times at 35-min intervals and once for 40 min after the end of the third stimulation.…”

Section: Electrical Field Stimulation (Efs)-induced Contraction Of Ramentioning

confidence: 99%

“…[1][2][3] Pharmacological studies using rats with xyleneirritated bladders, 4) cultured dorsal root ganglion (DRG) neurons, 5) and isolated bladder strips 6) suggest that KW-7158 has a unique mechanism of action, namely, it inhibits the contraction of the bladder smooth muscle by suppressing the activity of sensory nerves. This unique mode of action distinguishes KW-7158 as a drug candidate that may be free from the side effects caused by other anti-OAB drugs.…”

mentioning

confidence: 99%

“…6) To facilitate target identification, we have generated various DRG neuron-like cell lines, including TRD-10 cells, which express a large amount of the target, and TRD-49 cells, which express a negligible amount of the target. 6) In this study, using the DRG cell lines and a KW-7158 fluorescent derivative, we first screened the membrane protein expression library to identify clones expressing KW-7158 binding molecules. This resulted in the identification of a single clone that codes equilibrative nucleoside transporter-1 (ENT1).…”

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confidence: 99%

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The Anti-overactive Bladder Activity of KW-7158 Is Mediated by Blocking Equilibrative Nucleoside Transporter-1

Nishiya

Yamagata

Fukuda

et al. 2014

Biological & Pharmaceutical Bulletin

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KW-7158 is a novel therapeutic candidate for treating overactive bladder (OAB) with a unique mode of action: suppression of sensory afferent nerves. However, the molecular target of this compound remains unknown. We herein report the identification of the KW-7158 target to be equilibrative nucleoside transporter-1 (ENT1). A membrane protein expression library of ca. 7000 genes was expressed in a dorsal root ganglion cell line, which we had previously generated, and subjected to screening for binding with a fluorescent derivative that retains high binding activity to the target. The screening revealed that only cells transfected with an ENT1 expression vector exhibited significant binding. We next performed [ 3 H]KW-7158 binding experiments and an adenosine influx assay and found that KW-7158 binds to and inhibits ENT1. To further demonstrate the pharmacological relevance, we evaluated other known ENT1 inhibitors (nitrobenzylthioinosine, dipyridamole, draflazine) in an in vitro bladder strip contraction assay and the rat spinal cord injury OAB model. We found that all of the inhibitors exhibited anti-OAB activities, of which the potencies were comparable to that of adenosine influx inhibition in vitro. These studies demonstrated that the pharmacological target of KW-7158 is ENT1, at least in the rat OAB model. Our results will aid understanding of the precise mechanism of action of this drug and may also shed new light on the use of the adenosine pathway for the treatment of OAB.Key words nociceptive neuron; overactive bladder; adenosine transporter; drug target identification KW-7158 is a drug candidate for overactive bladder (OAB).1-3) Pharmacological studies using rats with xyleneirritated bladders, 4) cultured dorsal root ganglion (DRG) neurons, 5) and isolated bladder strips 6) suggest that KW-7158 has a unique mechanism of action, namely, it inhibits the contraction of the bladder smooth muscle by suppressing the activity of sensory nerves. This unique mode of action distinguishes KW-7158 as a drug candidate that may be free from the side effects caused by other anti-OAB drugs. However, the molecular target of KW-7158 remains elusive.We have previously shown that the target exists in the rat DRG using [ 3 H] KW-7158 radioligand binding assays. 6) To facilitate target identification, we have generated various DRG neuron-like cell lines, including TRD-10 cells, which express a large amount of the target, and TRD-49 cells, which express a negligible amount of the target. 6)In this study, using the DRG cell lines and a KW-7158 fluorescent derivative, we first screened the membrane protein expression library to identify clones expressing KW-7158 binding molecules. This resulted in the identification of a single clone that codes equilibrative nucleoside transporter-1 (ENT1). Further in vitro and in vivo pharmacological studies demonstrated that KW-7158 is a novel ENT1 inhibitor and that the inhibition of ENT1 counteracts overactive bladder in the rat spinal cord injury OAB model. MATERIALS AND METHODSReagents KW-...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Electrical Field Stimulation (Efs)-induced Contraction Of Ramentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Anti-overactive Bladder Activity of KW-7158 Is Mediated by Blocking Equilibrative Nucleoside Transporter-1

Nishiya

Yamagata

Fukuda

et al. 2014

Biological & Pharmaceutical Bulletin

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The NaV1.7 Channel Subtype as an Antinociceptive Target for Spider Toxins in Adult Dorsal Root Ganglia Neurons

et al. 2018

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Although necessary for human survival, pain may sometimes become pathologic if long-lasting and associated with alterations in its signaling pathway. Opioid painkillers are officially used to treat moderate to severe, and even mild, pain. However, the consequent strong and not so rare complications that occur, including addiction and overdose, combined with pain management costs, remain an important societal and economic concern. In this context, animal venom toxins represent an original source of antinociceptive peptides that mainly target ion channels (such as ASICs as well as TRP, CaV, KV and NaV channels) involved in pain transmission. The present review aims to highlight the NaV1.7 channel subtype as an antinociceptive target for spider toxins in adult dorsal root ganglia neurons. It will detail (i) the characteristics of these primary sensory neurons, the first ones in contact with pain stimulus and conveying the nociceptive message, (ii) the electrophysiological properties of the different NaV channel subtypes expressed in these neurons, with a particular attention on the NaV1.7 subtype, an antinociceptive target of choice that has been validated by human genetic evidence, and (iii) the features of spider venom toxins, shaped of inhibitory cysteine knot motif, that present high affinity for the NaV1.7 subtype associated with evidenced analgesic efficacy in animal models.

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The generation of rat dorsal root ganglion cell lines to identify the target of KW-7158, a novel treatment for overactive bladder

Cited by 2 publications

References 27 publications

The Anti-overactive Bladder Activity of KW-7158 Is Mediated by Blocking Equilibrative Nucleoside Transporter-1

The Anti-overactive Bladder Activity of KW-7158 Is Mediated by Blocking Equilibrative Nucleoside Transporter-1

The NaV1.7 Channel Subtype as an Antinociceptive Target for Spider Toxins in Adult Dorsal Root Ganglia Neurons

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