Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, PC09
Expression of N-arachidonoylethanolamine (anandamide)-synthetizing enzymes in rat primary sensory neurons
A. Varga1, A. Jenes2, L. Buluwela1, I. Nagy1
1. Dept. of Surgery and Cancer, Imperial College London, London, United Kingdom. 2. Dept. of Physiology, University of Debrecen, Debrecen, Hungary.
The endovanilloid/endocannabinoid systems control the activity and excitability of a major sub-population of nociceptive primary sensory neurons (PSN) (1), through the signalling molecule anandamide (AEA) and its main targets, the excitatory transient receptor potential vanilloid type 1 ion channel (TRPV1) and the inhibitory cannabinoid 1 (CB1) receptor (2; 3). AEA that intriguingly is also produced by PSN expressing TRPV1 and CB1 receptors, is thought to be synthesized by multiple enzymatic pathways (4). At present, the enzymatic pathway(s) involved in AEA synthesis in PSN is not known. Here, we aimed to find the expression pattern of enzymes, which might participate in AEA production in PSN. Dorsal root ganglia (DRG) were collected from male Sprague Dawley rats (80-100g). Total RNA and protein were isolated both from intact DRG and 2 days old cultures of PSN prepared from the DRG. Half of the cultures grew in the presence of capsaicin (10 μM) overnight to induce degeneration of the great majority of TRPV1-expressing PSN (5). Reverse-transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR (RT-qPCR) and western immunoblotting were used for studying enzyme expression. All data are expressed as mean ± SEM. Differences are regarded significant at p<0.05 calculated by Student's t-test. RT-PCR and RT-qPCR revealed that mRNA of all enzymes are expressed in intact DRG, though inositol 5’ phosphatase (SHIP-1) and protein tyrosine phosphatase non-receptor type 22 (PTPn22) mRNA levels were low. Culturing caused a significant down-regulation in the expression of alpha/beta-hydrolase 4 (ABHD4) and group 1B soluble phospholipase (sPLA2G1B) transcripts (0.18±0.01, p<0.01 and 0.8±0.03, p<0.05, respectively; n=4), whereas it elevated the expression of protein tyrosine phosphatase, non-receptor type 22 (PTPn22) transcript (2.75 ± 0.18 p<0.01) when compared to that of intact DRG. Capsaicin treatment resulted in significant increase in ABHD4 transcription (1.87 ± 0.25 p<0.01; n=4) in comparison to that of untreated cultures. Western-blotting revealed that N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), glycerophosphodiester phosphodiesterase 1 (GDE-1) and PTPn22 proteins are present in intact DRG. Culturing increased the level of these enzymes. In addition, sPLA2G1B became also detectable in cultures. Capsaicin treatment diminished the expression of NAPE-PLD and GDE-1 proteins (0.74±0.02 p<0.05 and 0.62±0.06 p<0.05, respectively; n=3). Our data indicate that several putative AEA-synthesising pathways could indeed be involved in AEA production in PSN. Among these pathways, NAPE-PLD and/or GDE-1 might produce AEA in TRPV1-expressing PSN.
Where applicable, experiments conform with Society ethical requirements