Deletion of ENTPD3 does not impair nucleotide hydrolysis in primary somatosensory neurons or spinal cord

Abstract

Ectonucleotidases are membrane-bound or secreted proteins that hydrolyze extracellular nucleotides. Recently, we identified three ectonucleotidases that hydrolyze extracellular adenosine 5’-monophosphate (AMP) to adenosine in primary somatosensory neurons. Currently, it is unclear which ectonucleotidases hydrolyze ATP and ADP in these neurons. Ectonucleoside triphosphate diphosphohydrolases (ENTPDs) comprise a class of enzymes that dephosphorylate extracellular ATP and ADP. Here, we found that ENTPD3 (also known as NTPDase3 or CD39L3) was located in nociceptive and non-nociceptive neurons of the dorsal root ganglion (DRG), in the dorsal horn of the spinal cord, and in free nerve endings in the skin. To determine if ENTPD3 contributes directly to ATP and ADP hydrolysis in these tissues, we generated and characterized an Entpd3 knockout mouse. This mouse lacks ENTPD3 protein in all tissues examined, including the DRG, spinal cord, skin, and bladder. However, DRG and spinal cord tissues from Entpd3 -/- mice showed no reduction in histochemical staining when ATP, ADP, AMP, or UTP were used as substrates. Additionally, using fast-scan cyclic voltammetry (FSCV), adenosine production was not impaired in the dorsal spinal cord of Entpd3 -/- mice when the substrate ADP was applied. Further, Entpd3 -/- mice did not differ in nociceptive behaviors when compared to wild-type mice, although Entpd3 -/- mice showed a modest reduction in β-alanine-mediated itch. Taken together, our data indicate that deletion of Entpd3 does not impair ATP or ADP hydrolysis in primary somatosensory neurons or in dorsal spinal cord. Moreover, our data suggest there could be multiple ectonucleotidases that act redundantly to hydrolyze nucleotides in these regions of the nervous system.

Neuronal NTPDase3 Mediates Extracellular ATP Degradation in Trigeminal Nociceptive Pathway

“…In this study, we extended this work to demonstrate that NTPDase3 is expressed in trigeminal ganglia nociceptive neurons and their axons. These results are consistent with NTPDase3 being detected in primary nociceptive neurons in the dorsal root ganglia [ 11 , 27 , 28 ] and trigeminal peripheral nerve Raschkow’s plexus in dental pulp [ 18 ]. We further confirmed functional ecto-ATPase activity in trigeminal ganglia neurons and associated nerve fibers that project to the brainstem and inhibition by anti-NTPDase3 serum.…”

“…By controlling the degradation of ATP within the synapse, NTPDase3 in presynaptic terminals is expected to reduce the amplitude and duration of pain signal transmission. However, NTPDase3 knock out mice display normal ecto-ATPase activity in the nociceptive lamina and do not show altered nociceptive behavior [ 27 ], suggesting that expression of alternative ecto-ATPases may be present or upregulated to compensate for the absence of NTPDase3 in the knock out mice. Interestingly, our ecto-ATPase activity blocking experiment showed that incubation with anti-NTPDase3 serum only partially reduced the ecto-ATPase activity in the nociceptive lamina.…”

Ectonucleoside triphosphate diphosphohydrolases and ecto-5′-nucleotidase in purinergic signaling: how the field developed and where we are now

“…The study proved its fundamental role in hemostasis and thrombosis. This was followed by the deletion of Entpd2 , the gene encoding NTPDase2, which allowed to analyze the function of the enzyme in taste buds [ 49 ], followed by the deletion of NTPDas3 [ 50 ]. Moreover, transgenic overexpression of NTPDase1 in mice or pigs permitted insight into its role in multiple organ systems.…”