Proceedings of The Physiological Society

King's College London (2011) Proc Physiol Soc 22, PC25

Poster Communications

Expression of N-acyl phosphotidylethanolamine phospholipase D in rat dorsal root ganglion neurons

J. Valente1,4, H. Tailor1, A. Jenes1, K. Mackie2, F. Cravatt3, L. Buluwala1, A. Avelino4, I. Nagy1

1. Department of Surgery and Cancer, Imperial College London, London, United Kingdom. 2. Centre of Biomolecular Sciences, Indiana University, Bloomington, Indiana, United States. 3. Department of Chemistry and Physiology, Scripps Institute, La Jolla, California, United States. 4. Institute of. Histology and Embryology, University of Porto, Porto, Portugal.

The enzyme N-acyl phosphotidylethanolamine phospholipase D (NAPE-PLD) is involved in Ca2+-dependent synthesis of anandamide (1) that is an endogenous activator of the transient receptor potential vanilloid type 1 ion channel (TRPV1) (2) and the cannabinoid 1 (CB1) receptor (3). TRPV1 and the CB1 receptor are involved in the regulation of the activity and excitability of a major sub-population of nociceptive primary sensory neurons (PSN) which play a pivotal role in the initiation and maintenance of acute pain as well as pain associated with pathological condition, such as inflammation (4). We have shown previously that TRPV1-expressing cells produce anandamide in a Ca2+-dependent manner. Further we have shown recently that NAPE-PLD mRNA is expressed in a sub-population of PSN and that most of the NAPE-PLD-expressing cells express TRPV1 (5). Here, we assessed the neurochemical properties of NAPE-PLD-expressing PSN. Multiple immunofluorescent staining was used on the L4 dorsal root ganglia (DRG) of naive, adult Wistar rats (n=3) or rats injected intraplantarly, under isoflurane anaesthesia (5% for induction and 3% for maintainance), with compelet Freund’s Adjuvant (CFA, n=3) or incomplete Freund’s Adjuvant (IFA; n=3) 3 days prior to tissue harvesting. We used anti- NAPE-PLD-, anti-neurofilament 200 (NG200)-; anti-calcitonin gene-related peptide (CGRP)- anti-CB1 receptor-, anti-TRPV1-antibodies and biotinylated Griffonia simplicifolia lectin (IB4). Immunoreactivity for NAPE-PLD was detected in ~40% of the cells. Most of them were immunonegative for NF200 that identifies cells with myelinated axons. Triple staining revealed that NAPE-PLD immunopositivity was present in cells expressing the nociceptive PSN markers, CGRP or IB4 binding sites. About 1/3 of the NAPE-PLD immunopositive cells expressed both CGRP immunopisitivity and IB4 binding sites. Further staining revealed that NAPE-PLD immunopositivity is highly co-expressed with immunopositivity for both of the anandamide-responding receptors, TRPV1 (~2/3), and the CB1 receptor (~90%). CFA but not IFA injection produced an overall increase in the number of NAPE-PLD immunopositive neurons (p<0.05, ANOVA). The increase was particularly apparent in large sized cells. Our results confirm recent data that NAPE-PLD is expressed predominantly in nociceptive cells. The high degree of co-expression between NAPE-PLD, TRPV1 and the CB1 receptor suggests that NAPE-PLD through synthesizing anandamide might play an important role in regulating the activity and excitability of nociceptive DRG neurons. The increase in NAPE-PLD expression following CFA injection in PSN neurons further suggests that that regulatory role could be enhanced in inflammatory conditions.

Where applicable, experiments conform with Society ethical requirements