Ectonucleotidases in the digestive system: focus on NTPDase3 localization

Abstract

Extracellular nucleotides and adenosine are biologically active molecules that bind members of the P2 and P1 receptor families, respectively. In the digestive system, these receptors modulate various functions, including salivary, gastric, and intestinal epithelial secretion and enteric neurotransmission. The availability of P1 and P2 ligands is modulated by ectonucleotidases, enzymes that hydrolyze extracellular nucleotides into nucleosides. Nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5'-nucleotidase are the dominant ectonucleotidases at physiological pH. While there is some information about the localization of ecto-5'-nucleotidase and NTPDase1 and -2, the distribution of NTPDase3 in the digestive system is unknown. We examined the localization of these ectonucleotidases, with a focus on NTPDase3, in the gastrointestinal tract and salivary glands. NTPDase1, -2, and -3 are responsible for ecto-ATPase activity in these tissues. Semiquantitative RT-PCR, immunohistochemistry, and in situ enzyme activity revealed the presence of NTPDase3 in some epithelial cells in serous acini of salivary glands and mucous acini and duct cells of sublingual salivary glands, in cells from the stratified esophageal and forestomach epithelia, and in some enteroendocrine cells of the gastric antrum. Interestingly, NTPDase2 and ecto-5'-nucleotidase are coexpressed with NTPDase3 in salivary gland cells and stratified epithelia. In the colon, neurons express NTPDase3 and glial cells express NTPDase2. Ca(2+) imaging experiments demonstrate that NTPDases regulate P2 receptor ligand availability in the enteric nervous system. In summary, the specific localization of NTPDase3 in the digestive system suggests functional roles of the enzyme, in association with NTPDase2 and ecto-5'-nucleotidase, in epithelial functions such as secretion and in enteric neurotransmission.

Cellular function and molecular structure of ecto-nucleotidases

“…They reveal overlapping tissue distributions but in situ they are mostly expressed by different cell types [35]. Examples of cellular co-expression of NTPDases identified by immunocytochemistry include epithelial cells in a variety of tissues [40]. NTPDase1 is the most thoroughly investigated enzyme and considerable insight has been gained from the generation of knockout mice [41,42].…”

“…Depending on mass action, a phosphate can be transferred from one ADP molecule to another, resulting in the formation of ATP and AMP (or vice versa) NTPDase2 is expressed on the adventitial surface of blood vessels where it contributes to vascular hemostasis [46,47]. It is co-expressed with NTPDase3 and eN in salivary cells and stratified epithelia of the gastrointestinal tract [40]. It is also found on taste buds [51] and was identified in a variety of tumor cells [37].…”

“…NTPDase3 is expressed in subsets of neurons in the brain expressing the neuropeptide hypocretin-1/orexin-A [59], in a variety of epithelia including kidney, airways, reproductive and digestive systems [40], and in all Langerhans islet Fig. 3 Radial phylogenetic tree of NTPDases.…”

Rat submandibular glands secrete nanovesicles with NTPDase and 5′-nucleotidase activities

“…We also analyzed the effects of time and NaN 3 on ATPase activity in SMG microsomes and observed again that at least two different NTPDases contributed to ATP and ADP hydrolysis. An unstable and azideresistant activity can be assigned to NTPDase2, like the human ecto-ATPase [31], and the stable and azide-sensible activity could be attributed mainly to NTPDase3, both coexpressed with ecto-5′-nucleotidase in mouse submandibular acini [14]. Western blots with specific antibodies indicate that NTPDase1, NTPDase2, and NTPDase3 and ecto-5′-nucleotidase are all present in the microsomal fraction (P27) and in the PM-enriched post-microsomal fraction (P100).…”

“…NTPDase1 was identified by immunohistochemistry in mucous acinar and ductal cells and also zymogen granules of mouse submandibular salivary gland [13]. NTPDase2 and ecto-5′-nucleotidase are co-expressed with NTPDase3 in mouse parotid and submandibular serous acinar cells but not in ductal cells [14]. Although all these enzymes are considered as PM-anchored proteins, the expression of NTPDase1, NTPDase2, and NTPDase3 has been also described in Golgi and endoplasmic reticulum (ER) membranes [15][16][17][18].…”