Na-K-ATPase in lacrimal gland acinar cell endosomal system: correcting a case of mistaken identity

Abstract

Na-K-ATPase is associated with a variety of membrane populations in lacrimal acinar cells. Acinus-like structures formed by rabbit acinar cells in primary culture were incubated with horseradish peroxidase (HRP) to label basolateral and endosomal membranes and then analyzed by electron microscopy cytochemistry with the 3-3'-diaminobenzidine reaction or by fractionation and measurement of marker catalytic activities or immunoreactivities. HRP adsorbed to basolateral membranes at 4 degrees C. Fractionation showed it associated with low-density membranes enriched in acid phosphatase and TGN38 but containing only minor amounts of Na-K-ATPase. Cells internalized HRP to cytoplasmic vesicles, Golgi structures, and lysosomes at 37 degrees C. The major endosomal compartment revealed by fractionation coincided with major peaks of Na-K-ATPase and Rab6 and secondary peaks of galactosyltransferase and gamma-adaptin. Carbachol (10 microM) increased lysosomal and Golgi labeling. Thus most of the Na-K-ATPase is located in the basolateral membrane-oriented endosomal system, concentrated in a compartment possibly related to the trans-Golgi network. Constitutive and stimulation-accelerated traffic to and from this compartment may serve several exocrine cell functions.

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

“…All animal procedures were in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication no. 85-23, revised 1996) (9,17,18) and were approved by the University of Southern California Institutional Animal Care and Use Committee. Acinar cells isolated from LG from New Zealand White rabbits (1.8 -2.2 kg) (Irish Farms, Norco, CA) were sequentially incubated in Mg 2ϩ -and Ca 2ϩ -free HBSS supplemented with EDTA and Ham's medium supplemented with collagenase, hyaluronidase, and DNAse, and cultured for 2 days in Peter's serum-free culture medium (18).…”

“…85-23, revised 1996) (9,17,18) and were approved by the University of Southern California Institutional Animal Care and Use Committee. Acinar cells isolated from LG from New Zealand White rabbits (1.8 -2.2 kg) (Irish Farms, Norco, CA) were sequentially incubated in Mg 2ϩ -and Ca 2ϩ -free HBSS supplemented with EDTA and Ham's medium supplemented with collagenase, hyaluronidase, and DNAse, and cultured for 2 days in Peter's serum-free culture medium (18). These cultured cells reform acinuslike structures displaying distinct apical and basolateral domains (9,17,24).…”

Biochemical Changes Contributing to Functional Quiescence in Lacrimal Gland Acinar Cells after Chronic Ex Vivo Exposure to a Muscarinic Agonist

“…L-quinuclidinylphenyl-4-3 H-benzilate ([ 3 H]-QNB) was obtained from Amersham (Arlington Heights, IL, USA). All other reagents were from standard suppliers as detailed in previous publications [19,[21][22][23][24][25].…”

“…Subcellular analysis. Control and carbachol-treated cell samples were harvested, washed, resuspended, lysed and analysed by isopycnic centrifugation in preformed sorbitol density gradients as described in previous studies [19,[21][22][23][24][25]. Membrane pellets from the 12 resulting fractions as well as the pellets that formed beneath the gradients were resuspended in 1 ml aliquots of 5% sorbitol lysis buffer and snap frozen in liquid nitrogen, then stored at À80 C for later use.…”

“…Analytical methods. Protein and biochemical markers were determined with methods that have been described in detail elsewhere [19,[21][22][23]. MAChR ligand-binding activity was determined with [ 3 H]-QNB as described by Bradley et al [18].…”