Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules

Abstract

nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules. Am J Physiol Cell Physiol 304: C976 -C984, 2013. First published March 6, 2013; doi:10.1152/ajpcell.00371.2012.-Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca 2ϩ -regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.VNUT; SLC17A9; mucin granules; ATP release; mucin secretion EXTRACELLULAR NUCLEOTIDES are key components of the mucociliary clearance process that traps and removes inhaled particles and microorganisms from the lung. Nucleotides/nucleosides within the airway surface liquid (ASL) promote mucin secretion via activation of P2Y 2 receptors in mucous (goblet) cells and stimulate fluid secretion/mucin hydration via A 2B and P2Y 2 receptor activation in ciliated cells (11). Deficient fluid secretion results in abnormal mucous hydration/clearance, leading to lung failure in cystic fibrosis, the most prevalent potentially lethal genetic disease in the United States and a major factor contributing to the progressive airway obstruction associated with chronic obstructive lung disease (4).Despite the importance of ASL nucleotides in airway physiology, the mechanisms by which airway epithelial cells release nucleotides have just begun to be addressed. For example, we recently demonstrated that plasma membrane pannexin 1 largely contributes to conductive ATP release from normal airways dominated by ciliated cells (22). Deletion of the pannexin 1 gene resulted in impaired ATP release in mouse tracheas ex vivo (22).We also ...

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

“…1 and 2), confirms our early studies and validity of using such fluorescent markers [4]. VNUT and [10][11][12][13][14][15]. In freshly isolated pancreatic acini, secretory/zymogen granule marker Rab3D co-localizes with VNUT ( Fig.…”

“…Soon after, we provided evidence that VNUT is expressed in pancreatic zymogen granules and is engaged in ATP uptake [9]. Since then, expression of VNUT has been demonstrated in several cell types indicating the general importance of VNUT for ATP release [10][11][12][13][14][15].…”

“…Rat acinar cell line AR42J (CRL-1492, ATCC, passages [15][16][17][18][19][20][21][22][23][24][25] was cultured in RPMI media supplemented with 10 % fetal bovine serum (FBS) and penicillin/streptomycin. Cells were cultured in humidified incubators at 37°C, with 5 % CO 2 in air.…”

Expression of Vesicular Nucleotide Transporter in the Mouse Retina

“…17,18) VNUT is present in various ATP-storing secretory vesicles, such as adrenal chromaffin granules, insulin granules of islet β cells, glutamate-containing synaptic vesicles of hippocampal neurons, platelet granules, airway epithelial mucin granules, and Glucagon-like peptide-1 (GLP-1)-containing granules of intestinal L cells. 17,[19][20][21][22][23][24] VNUT −/− mice were shown to lose vesicular ATP content and vesicular ATP release, resulting in dissection of purinergic chemical transmission in vivo. 19) Thus, VNUT is primarily responsible for vesicular ATP storage and release, and plays an essential role in purinergic chemical transmission.…”

Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells

“…Exocytotic mechanisms involve ATP storage and Ca 2+ -regulated exocytotic release and SLC17A9 protein has been identified as a vesicular nucleotide transporter (VNUT) that plays a key role in ATP exocytosis [13]. VNUT expression has been detected in different types of cells [13][14][15][16][17][18][19]; however, its presence in corneal and conjunctival epithelial cells remains elusive.…”