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Capsaicin-sensitive primary sensory neurons in the mouse express N-Acyl phosphatidylethanolamine phospholipase D.

Nagy B, Fedonidis C, Photiou A, Wahba J, Paule CC, Ma D, Buluwela L, Nagy I - Neuroscience (2009)

Bottom Line: Furthermore, we also report that capsaicin application downregulates the expression of NAPE-PLD as well as the capsaicin receptor, transient receptor potential vanilloid type 1 ion channel, by about 70% in the cultures prepared from dorsal root ganglia.These findings indicate that a major sub-population of capsaicin-sensitive primary sensory neurons expresses NAPE-PLD, and suggest that NAPE-PLD is expressed predominantly by capsaicin-sensitive neurons in dorsal root ganglia.These data also suggest that NAPE-PLD might be a target to control the activity and excitability of a major sub-population of nociceptive primary sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, Faculty of Medicine, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK.

ABSTRACT
Our previous finding, that the capsaicin- and KCl-induced Ca(2+)-dependent production of the intra- and intercellular signaling molecule N-arachidonoyl ethanolamine (anandamide) in cultured primary sensory neurons could be abolished and reduced by approximately 2/3 by capsaicin-induced degeneration of capsaicin-sensitive neurons, respectively suggests that a major sub-population of capsaicin-sensitive cells together with a group of non-capsaicin-sensitive cells should express enzymes involved in Ca(2+)-dependent anandamide synthesis. N-acyl phosphotidylethanolamine phospholipase D (NAPE-PLD) is known to be involved in Ca(2+)-dependent anandamide production. Hence, here, we used reverse transcriptase and quantitative real time polymerase chain reaction to study NAPE-PLD expression in dorsal root ganglia and to clarify the sub-population of cells expressing this enzyme. Cultures prepared from mouse dorsal root ganglia were grown either in the absence or presence of the neurotoxin, capsaicin (10 muM) overnight. We report, that NAPE-PLD is expressed both in dorsal root ganglia and cultures prepared from dorsal root ganglia and grown in the absence of capsaicin. Furthermore, we also report that capsaicin application downregulates the expression of NAPE-PLD as well as the capsaicin receptor, transient receptor potential vanilloid type 1 ion channel, by about 70% in the cultures prepared from dorsal root ganglia. These findings indicate that a major sub-population of capsaicin-sensitive primary sensory neurons expresses NAPE-PLD, and suggest that NAPE-PLD is expressed predominantly by capsaicin-sensitive neurons in dorsal root ganglia. These data also suggest that NAPE-PLD might be a target to control the activity and excitability of a major sub-population of nociceptive primary sensory neurons.

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RT-PCR analysis of NAPE-PLD gene expression. (A) Agarose gel electrophoresis of RT-PCR products for GAPDH (370 bp) from cDNA made to RNA from brain, heart, DRG and cultures prepared from DRG (lanes 1, 2, 3, and 4, respectively). (B) Agarose gel electrophoresis of RT-PCR products for NAPE-PLD (222 bp) from cDNA made to RNA from brain, heart, DRG and cultures prepared from DRG (lanes 1, 2, 3, and 4, respectively). Note that NAPE-PLD is expressed in all the tissues we examined. (C) NAPE-PLD (lane 1) and GAPDH (lane 4) gene expression in mouse dorsal root ganglion cultures grown under control conditions (without capsaicin). Lane 2 shows NAPE-PLD expression in a brain sample collected from the same animal used to derive the cultures analyzed in lanes 1 and 4. Lane 3 is a control PCR reaction, where RNA equivalent to the cDNA used in lane 1 has been used as template. (D) NAPE-PLD (lane 1) and GAPDH (lane 4) gene expression in mouse dorsal root ganglion cultures grown in the presence of 10 μM capsaicin overnight. Lane 2 shows NAPE-PLD expression in the brain sample collected from the same animal. Lane 3 is a control PCR reaction, where RNA equivalent to the cDNA used in lane 1 has been used as template. Note that the treatment of cultures with capsaicin significantly downregulated NAPE-PLD expression (lane 1 in C and D.)
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fig1: RT-PCR analysis of NAPE-PLD gene expression. (A) Agarose gel electrophoresis of RT-PCR products for GAPDH (370 bp) from cDNA made to RNA from brain, heart, DRG and cultures prepared from DRG (lanes 1, 2, 3, and 4, respectively). (B) Agarose gel electrophoresis of RT-PCR products for NAPE-PLD (222 bp) from cDNA made to RNA from brain, heart, DRG and cultures prepared from DRG (lanes 1, 2, 3, and 4, respectively). Note that NAPE-PLD is expressed in all the tissues we examined. (C) NAPE-PLD (lane 1) and GAPDH (lane 4) gene expression in mouse dorsal root ganglion cultures grown under control conditions (without capsaicin). Lane 2 shows NAPE-PLD expression in a brain sample collected from the same animal used to derive the cultures analyzed in lanes 1 and 4. Lane 3 is a control PCR reaction, where RNA equivalent to the cDNA used in lane 1 has been used as template. (D) NAPE-PLD (lane 1) and GAPDH (lane 4) gene expression in mouse dorsal root ganglion cultures grown in the presence of 10 μM capsaicin overnight. Lane 2 shows NAPE-PLD expression in the brain sample collected from the same animal. Lane 3 is a control PCR reaction, where RNA equivalent to the cDNA used in lane 1 has been used as template. Note that the treatment of cultures with capsaicin significantly downregulated NAPE-PLD expression (lane 1 in C and D.)

Mentions: RT-PCR produced distinct products with sizes between 350 and 400 bp, and 200 and 250 bp in all samples (Fig. 1A, B). While the larger product (Fig. 1A) corresponded with the predicted size of the GAPDH, the smaller product (Fig. 1B) corresponded with that expected for the NAPE-PLD RT-PCR product. These findings indicated that NAPE-PLD is expressed both in DRG and cultures prepared from DRG.


Capsaicin-sensitive primary sensory neurons in the mouse express N-Acyl phosphatidylethanolamine phospholipase D.

Nagy B, Fedonidis C, Photiou A, Wahba J, Paule CC, Ma D, Buluwela L, Nagy I - Neuroscience (2009)

RT-PCR analysis of NAPE-PLD gene expression. (A) Agarose gel electrophoresis of RT-PCR products for GAPDH (370 bp) from cDNA made to RNA from brain, heart, DRG and cultures prepared from DRG (lanes 1, 2, 3, and 4, respectively). (B) Agarose gel electrophoresis of RT-PCR products for NAPE-PLD (222 bp) from cDNA made to RNA from brain, heart, DRG and cultures prepared from DRG (lanes 1, 2, 3, and 4, respectively). Note that NAPE-PLD is expressed in all the tissues we examined. (C) NAPE-PLD (lane 1) and GAPDH (lane 4) gene expression in mouse dorsal root ganglion cultures grown under control conditions (without capsaicin). Lane 2 shows NAPE-PLD expression in a brain sample collected from the same animal used to derive the cultures analyzed in lanes 1 and 4. Lane 3 is a control PCR reaction, where RNA equivalent to the cDNA used in lane 1 has been used as template. (D) NAPE-PLD (lane 1) and GAPDH (lane 4) gene expression in mouse dorsal root ganglion cultures grown in the presence of 10 μM capsaicin overnight. Lane 2 shows NAPE-PLD expression in the brain sample collected from the same animal. Lane 3 is a control PCR reaction, where RNA equivalent to the cDNA used in lane 1 has been used as template. Note that the treatment of cultures with capsaicin significantly downregulated NAPE-PLD expression (lane 1 in C and D.)
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724038&req=5

fig1: RT-PCR analysis of NAPE-PLD gene expression. (A) Agarose gel electrophoresis of RT-PCR products for GAPDH (370 bp) from cDNA made to RNA from brain, heart, DRG and cultures prepared from DRG (lanes 1, 2, 3, and 4, respectively). (B) Agarose gel electrophoresis of RT-PCR products for NAPE-PLD (222 bp) from cDNA made to RNA from brain, heart, DRG and cultures prepared from DRG (lanes 1, 2, 3, and 4, respectively). Note that NAPE-PLD is expressed in all the tissues we examined. (C) NAPE-PLD (lane 1) and GAPDH (lane 4) gene expression in mouse dorsal root ganglion cultures grown under control conditions (without capsaicin). Lane 2 shows NAPE-PLD expression in a brain sample collected from the same animal used to derive the cultures analyzed in lanes 1 and 4. Lane 3 is a control PCR reaction, where RNA equivalent to the cDNA used in lane 1 has been used as template. (D) NAPE-PLD (lane 1) and GAPDH (lane 4) gene expression in mouse dorsal root ganglion cultures grown in the presence of 10 μM capsaicin overnight. Lane 2 shows NAPE-PLD expression in the brain sample collected from the same animal. Lane 3 is a control PCR reaction, where RNA equivalent to the cDNA used in lane 1 has been used as template. Note that the treatment of cultures with capsaicin significantly downregulated NAPE-PLD expression (lane 1 in C and D.)
Mentions: RT-PCR produced distinct products with sizes between 350 and 400 bp, and 200 and 250 bp in all samples (Fig. 1A, B). While the larger product (Fig. 1A) corresponded with the predicted size of the GAPDH, the smaller product (Fig. 1B) corresponded with that expected for the NAPE-PLD RT-PCR product. These findings indicated that NAPE-PLD is expressed both in DRG and cultures prepared from DRG.

Bottom Line: Furthermore, we also report that capsaicin application downregulates the expression of NAPE-PLD as well as the capsaicin receptor, transient receptor potential vanilloid type 1 ion channel, by about 70% in the cultures prepared from dorsal root ganglia.These findings indicate that a major sub-population of capsaicin-sensitive primary sensory neurons expresses NAPE-PLD, and suggest that NAPE-PLD is expressed predominantly by capsaicin-sensitive neurons in dorsal root ganglia.These data also suggest that NAPE-PLD might be a target to control the activity and excitability of a major sub-population of nociceptive primary sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, Faculty of Medicine, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK.

ABSTRACT
Our previous finding, that the capsaicin- and KCl-induced Ca(2+)-dependent production of the intra- and intercellular signaling molecule N-arachidonoyl ethanolamine (anandamide) in cultured primary sensory neurons could be abolished and reduced by approximately 2/3 by capsaicin-induced degeneration of capsaicin-sensitive neurons, respectively suggests that a major sub-population of capsaicin-sensitive cells together with a group of non-capsaicin-sensitive cells should express enzymes involved in Ca(2+)-dependent anandamide synthesis. N-acyl phosphotidylethanolamine phospholipase D (NAPE-PLD) is known to be involved in Ca(2+)-dependent anandamide production. Hence, here, we used reverse transcriptase and quantitative real time polymerase chain reaction to study NAPE-PLD expression in dorsal root ganglia and to clarify the sub-population of cells expressing this enzyme. Cultures prepared from mouse dorsal root ganglia were grown either in the absence or presence of the neurotoxin, capsaicin (10 muM) overnight. We report, that NAPE-PLD is expressed both in dorsal root ganglia and cultures prepared from dorsal root ganglia and grown in the absence of capsaicin. Furthermore, we also report that capsaicin application downregulates the expression of NAPE-PLD as well as the capsaicin receptor, transient receptor potential vanilloid type 1 ion channel, by about 70% in the cultures prepared from dorsal root ganglia. These findings indicate that a major sub-population of capsaicin-sensitive primary sensory neurons expresses NAPE-PLD, and suggest that NAPE-PLD is expressed predominantly by capsaicin-sensitive neurons in dorsal root ganglia. These data also suggest that NAPE-PLD might be a target to control the activity and excitability of a major sub-population of nociceptive primary sensory neurons.

Show MeSH
Related in: MedlinePlus