NA+-K+-ATPase Pump (NKAp) Activity  in alveolar type II cells increases with stretch

NKAp’s in the alveolar epithelium (AE) create a transepithelial Na+ gradient crucial to keeping the airspace fluid-free. We hypothesize that stretch of the AE upregulates NKAp activity.  Alveolar Type II epithelial cells were isolated from Sprague-Dawley rats (n=21) and seeded onto fibronectin-coated Silastic membranes mounted in small wells.  After two days in culture, cells were cyclically, biaxially stretched in a custom-made stretching device for 1 hr.  After stretch, cells were bathed for 5 minutes in a physiological medium containing 86Rb+, a K+-channel tracer commonly used to monitor NKAp activity.  Half of the wells were pretreated with ouabain to inhibit NKAp activity.  Cells were then rinsed (5x) with an ice-cold, Ba2+-containing solution, to trap all absorbed 86Rb+ inside the cells and prevent further uptake.  After air drying, cells were solubilized, and samples were taken from each well for scintillation counting and protein assay.  Observations were expressed in counts/min/µg protein. NKAp activity was reported as the difference in 86Rb+ uptake between wells treated or not treated with ouabain.  NKAp activity was further compared to unstretched controls. Results supported our hypothesis, indicating statistically significant increases in pump activity for cells stretched at 12, 25, and 37% change in surface area (DSA), which relates to 60, 80, and 100% TLC, respectively.  In contrast, comparative studies with A549 cells, a transformed Type II cell line, showed no significant change in pump activity at  25% DSA.  Also notable, cells treated with gadolinium, a non-specific stretch-activated channel (SAC) blocker, showed significant increases in NKAp activity, but significantly less than increases observed in non-gadolinium-treated cells.  These data suggest both a primary mechanocouple between cell stretch and NKAp activity, and a secondary SAC-modulated pathway.