Dr. Eric Lingueglia and his team at LabEx Ion Channel Science and Therapeutics, 06560 Valbonne, France continue to do excellent work using our i-Fect siRNA Transfection Kit. Here they use the kit to help validate the potential of a new class of three-finger peptides from the black mamba to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons: Sylvie Diochot, Anne Baron, Miguel Salinas, Dominique Douguet, Sabine Scarzello, Anne-Sophie Dabert-Gay, Delphine Debayle, Valérie Friend, Abdelkrim Alloui, Michel Lazdunski, Eric Lingueglia. Black mamba venom peptides target acid-sensing ion channels to abolish pain. Nature (2012)doi:10.1038/nature11494.
...Locally designed siRNAs targeting ASIC1 (si-ASIC1a/1b, GCCAAGAAGUUCAACAAAUdtdt), ASIC2 (si-ASIC2a/2b, UGAUCAAAGAGAAGCUAUUdtdt) and ASIC2a (si-ASIC2a, AGGCCAACUUCAAACACUAdtdt) have been validated in vitro in COS-7 cells transfected with myc-ASIC1a, ASIC1b, myc-ASIC2a or myc-ASIC2b, and the relevant siRNA or a control siRNA (si-CTR, GCUCACACUACGCAGAGAUdtdt) with TransIT-LT1 and transIT-TKO (Mirus), respectively. Cells were lysed 48 h after transfection and processed for western blot analysis to assess the amount of ASIC1a protein with the anti-Myc A14 antibody (1:500; Santa Cruz Biotechnology) or the anti-ASIC1 antibody (N271/44; 1:300; NeuroMab) and a monoclonal antibody against actin (AC-40; 1:1,000; Sigma) as a loading control. siRNAs were intrathecally injected into mice (2 µg per mouse at a ratio of 1:4 (w/v) with i-Fect (Neuromics)) twice a day for 3 days. After 3 days of treatment, the paw-flick latency was measured and the residual effect of mambalgin-1 (intrathecal or intraplantar, 34 µM) or the carrageenan (intraplantar, 2%)-induced hyperalgesia was tested. For validation of the in vivo effect of the siRNAs, lumbar DRGs or lumbar dorsal spinal cord were removed after the last siRNA injection for total RNA isolation (RNeasy kits, Qiagen) followed by cDNA synthesis (AMV First-Strand cDNA synthesis kit (Invitrogen) and High Capacity RNA-to-cDNA Kit, (Applied Biosystems)). The relative amounts of ASIC transcripts were evaluated by quantitative reverse-transcription PCR in a Light-Cycler 480 (Roche Products). Pre-designed and validated TaqMan assays for ASIC1 (ASIC1a and ASIC1b; Mm01305998_mH), ASIC1a (Mm01305996_m1), ASIC2 (ASIC2a and ASIC2b; Mm00475691_m1), ASIC3 (Mm00805460_m1) and 18S ribosomal RNA (Mm03928990_g1) were from Applied Biosystems. Each cDNA sample was run in triplicate and results were normalized against 18S and converted to fold induction relative to control siRNA treatment...
Showing posts with label ACID-sensing ion channels. Show all posts
Showing posts with label ACID-sensing ion channels. Show all posts
Sunday, October 21, 2012
Monday, April 25, 2011
ASICs and Surgical Pain
Dr. Eric Lingueglia, an INSERM group leader, and his team at the CNRS IPMC/IN2M have been doing impressive research using our i-Fect ™ siRNA Transfection Kits to study the role of Acid-Sensing Ion Channels in Postoperative Pain.
The etiology and pathophysiology of this pain is poorly understood. Their work is shedding light on potential root causes:
Emmanuel Deval, Jacques Noël, Xavier Gasull1, Anne Delaunay, Abdelkrim Alloui, Valérie Friend, Alain Eschalier, Michel Lazdunski, and Eric Lingueglia. Acid-Sensing Ion Channels in Postoperative Pain. The Journal of Neuroscience, 20 April 2011, 31(16): 6059-6066; doi: 10.1523/JNEUROSCI.5266-10.2011.
...Ten microliters of a siRNA (2 μg)/i-Fect (Neuromics) mix was injected intrathecally between the L4 and L5 vertebrae of rats using a Hamilton syringe and a 25 gauge needle. Animals received one injection per day for 4 d (Fig. 4A, protocol). ASIC3 (CUACACGCUAUGCCAAGGAdtdt) and the corresponding scramble (GCUCACACUACGCAGAGAUdtdt) siRNAs have been previously described (Deval et al., 2008)...
Highlights: Pharmacological inhibition of ASIC3 channels with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by small interfering RNA led to a significant reduction of postoperative spontaneous, thermal, and postural pain behaviors (spontaneous flinching, heat hyperalgesia, and weight bearing). ASIC3 appears to have an important role in deep tissue but also affects prolonged pain evoked by skin incision alone.
ASIC3s are excitatory ion channels directly activated by extracellular protons that detect the painful drops in pH at incision points. Several factors may participate in the drop of extracellular pH, such as release of the acidic content of lyzed cells, degranulation of mast cells, organic acids released by metabolism..etc This makes makes the Ion Channel a great marker for the studying activation of pain and a potential therapeutic target for mitigating surgical pain.
I will continue to track and report progress.
The etiology and pathophysiology of this pain is poorly understood. Their work is shedding light on potential root causes:
Emmanuel Deval, Jacques Noël, Xavier Gasull1, Anne Delaunay, Abdelkrim Alloui, Valérie Friend, Alain Eschalier, Michel Lazdunski, and Eric Lingueglia. Acid-Sensing Ion Channels in Postoperative Pain. The Journal of Neuroscience, 20 April 2011, 31(16): 6059-6066; doi: 10.1523/JNEUROSCI.5266-10.2011.
...Ten microliters of a siRNA (2 μg)/i-Fect (Neuromics) mix was injected intrathecally between the L4 and L5 vertebrae of rats using a Hamilton syringe and a 25 gauge needle. Animals received one injection per day for 4 d (Fig. 4A, protocol). ASIC3 (CUACACGCUAUGCCAAGGAdtdt) and the corresponding scramble (GCUCACACUACGCAGAGAUdtdt) siRNAs have been previously described (Deval et al., 2008)...
Highlights: Pharmacological inhibition of ASIC3 channels with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by small interfering RNA led to a significant reduction of postoperative spontaneous, thermal, and postural pain behaviors (spontaneous flinching, heat hyperalgesia, and weight bearing). ASIC3 appears to have an important role in deep tissue but also affects prolonged pain evoked by skin incision alone.
ASIC3s are excitatory ion channels directly activated by extracellular protons that detect the painful drops in pH at incision points. Several factors may participate in the drop of extracellular pH, such as release of the acidic content of lyzed cells, degranulation of mast cells, organic acids released by metabolism..etc This makes makes the Ion Channel a great marker for the studying activation of pain and a potential therapeutic target for mitigating surgical pain.
I will continue to track and report progress.
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