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Edit Toth-Molnar, Mate Katona, Eszter Vizvari, Ferenc Rarosi, Peter Orvos, Andrea Facsko, Viktoria Venglovecz, Zoltan Rakonczay, Chuanqing Ding, Peter Hegyi; Na+-K+-2Cl- Cotransporter in Rabbit Lacrimal Gland Ducts Plays a Key Role in Lacrimal Secretion. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2482.
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We recently reported that isolated duct segment from rabbit lacrimal gland (LG) was able to secrete into the luminal space in response to secretagogues, which was completely blocked by bumetanide, suggesting the presence of Cl- transport in these ducts, especially those mediated by Na+-K+-2Cl- cotransporter (NKCC1). While NKCC1 was present in the basolateral membranes of duct cells from rabbit LG, its role remains unclear, and therefore, the aim of the present work was to further investigate its activity in these ducts.
Immunofluorescence was used to confirm the localization of NKCC1. Rabbit LG interlobular ducts were isolated as we described before. Fluorophotometry with NH4+-pulse technique was used to elicit pH changes, and the rate of bumetanide-sensitive cytosolic acidification after addition of NH4+ was used to quantify the activity of NKCC1. Results were expressed as initial change of intracellular pH rate calculated for 30 and 60 seconds. A mixed ANOVA model was applied and p<0.05 was considered as significantly different.
Immunofluorescence confirmed the presence of NKCC1 in basolateral membranes of duct cells. While the basal activity of NKCC1 was minimally detectable (0.009±0.006 pH unit/30 sec and 0.013±0.010 pH unit/60 sec), addition of forskolin (10 μmol/L) caused a significant increase of its activity to 0.023±0.009 pH unit/30 sec (p=0.029) and 0.024±0.008 pH unit/60 sec (p=0.045). However, lower cytosolic [Cl-] had no significant effect on NKCC1 activity in the first 30 sec (0.008±0.003 pH unit/30 sec, p=0.071), whereas stimulation at 60 sec appeared to be borderline (0.020±0.007 pH unit/60 sec, p=0.053). Hyperosmotic challenge (390 mOsm) increased NKCC1 activity significantly at both 30 sec (0.023±0.003 pH unit, p=0.0001) and 60 sec (0.024±0.007 pH unit, p=0.025). Carbachol (100 μmol/L) had no significant effect on NKCC1 activity at either 30 sec (0.001±0.004 pH unit, p=0.867) or 60 sec (0.006±0.006 pH unit, p=0.388).<br /> <br />
Data presented here demonstrated the transmembrane ionic transport of duct epithelial cells, particularly the central role of Cl- transport in these cells, which is in part mediated by NKCC1. These results highlighted the functional involvement of NKCC1 and the pivotal role it may play in LG duct secretion, supporting the notion that these ducts play a key role in tear production.
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