The CF Salt Controversy R Tarran, B.R Grubb, D Parsons, M Picher, A.J Hirsh, C.W Davis, R.C Boucher  Molecular Cell  Volume 8, Issue 1, Pages 149-158 (July 2001) DOI: 10.1016/S1097-2765(01)00286-6

Figure 1 Wild-type (WT) and CF Murine Nasal Epithelial Morphology and Airway Surface Liquid (ASL) Height and [Cl−] In Vivo (A) Light micrographs depicting nasal epithelial surface cell morphology in WT (top) and CF (bottom) mice following in vivo fixation with OsO4 and PFC. Scale bars are 10 μm. (B) Summary data of goblet cell frequency in WTs and CFs. N = 6 (WT) and 7 (CF). (C) Transmission electron micrographs of ASL from WT (left) and CF (right) nasal epithelia following in vivo fixation with OsO4 and PFC. All scale bars are 4 μm. (D) Summary data of ASL height in WT (n = 4) and CF (n = 8) murine nasal epithelia. (E) Summary data for ASL [Cl−] measured by microdialysis for WT (n = 6) and CF (n = 3) mice. All data shown as mean ± SEM. * denotes p < 0.01 as compared to WTs Molecular Cell 2001 8, 149-158DOI: (10.1016/S1097-2765(01)00286-6)

Figure 2 The Effect of Salt (Ionic Osmolytes) on ASL Height and Composition in Human Normal Airway Cultures (A) Confocal images of cells (green) and ASL (red) obtained before, 2, and 12 min after mucosal addition of powdered NaCl (0.78 mg) suspended in PFC. (B) Plots of ASL height over time following NaCl addition (black lines; n = 8), KCl addition (blue lines; n = 8) and following amiloride and NaCl addition (red lines; n = 7). All data shown as mean ± SEM. * denotes p < 0.05 as compared to t = 0 for each subset. (C) ASL [Cl−] before (t = 0) and after NaCl addition, measured with double-barreled microelectrodes (n = 8) Molecular Cell 2001 8, 149-158DOI: (10.1016/S1097-2765(01)00286-6)

Figure 3 The Effect of Raffinose (Nonionic Osmolyte) on ASL Height and [Cl−] in Human Normal and CF Airway Cultures (A) Confocal images of cells and ASL obtained before, 10 min, and 4 hr after mucosal addition of powdered raffinose (8.0 mg) suspended in PFC. (B) Plots of ASL height following addition of raffinose to normal (♦) or CF (▴) ASL. N = 7 (normal) and 6 (CF). (C) [Cl−] measured with double-barreled microelectrodes following addition of raffinose to normal (♦) or CF (▴) ASL. N = 6 (normal) and 4 (CF). All data shown as mean ± SEM. * denotes p < 0.05 as compared to t = 0 for each subset. Data were not significantly different between normal and CF Molecular Cell 2001 8, 149-158DOI: (10.1016/S1097-2765(01)00286-6)

Figure 4 Responses of ASL from Human Cultures to Osmolytes over Prolonged (24 hr) Intervals (Ai) Time course of ASL volume on normal airway epithelia after apical addition of 50 μl test buffer at t = 0. Open bars, KBR (298 mOsM); solid bars, hypertonic saline (HS; 436 mOsM); hatched bars, raffinose (431 mosM). N = 20, 20, and 18 for KBR at t = 0, 3, and 24 hr, respectively; n = 15, 15, and 10 for HS at t = 0, 3, and 24 hr, respectively; and for raffinose n = 17, 17, and 14 for t = 0, 3, and 24 hr respectively. * p ≤ 0.05 from the KBR values at the same time points. + p < 0.05 from respective treatments at t = 3 hr. (Aii) Osmolality of ASL for normal airway cultures measured at the times indicated. KBR, n = 3; HS, n = 3; and raffinose, n = 5. * = p < 0.05 from KBR. (Aiii) Cl− concentration in the ASL from normal airway cultures measured at the times indicated. KBR, n = 3 at each time point; HS, n = 3 at each time point; for raffinose, n = 5 for each time point. * p ≤ 0.05 from KBR values at same time point. (B) The effect of apical amiloride on ASL volume after 24 hr, as compared to nonamiloride controls from Ai. Open bars, ASL volume; solid bars, ASL osmolality; hatched bars, ASL [Cl−]. KBR + amiloride, n = 8; HS + amiloride, n = 10; raffinose + amiloride, n = 4. * p ≤ 0.05 no amiloride vs. amiloride pretreatment for each respective osmolyte. The HS and raffinose preparations treated with amiloride contained significantly more volume (p ≤ 0.05) than the KBR preparations treated with amiloride. (C) ASL remaining on CF cultures 24 hr after addition of indicated solutions. Dashed line indicates volume at t = 0. The raffinose dosed cultures contained significantly more ASL than cultures incubated in apical KBR or hypertonic NaCl (* p < 0.05). + p < 0.05 different to normal cultures from (A). N = 8 for all treatments. Bars are means ± SEM Molecular Cell 2001 8, 149-158DOI: (10.1016/S1097-2765(01)00286-6)

Figure 5 The Effect of UTP on ASL Height and Composition in Human Normal and CF Airway Cultures (A) Confocal images of ASL obtained before and 10 min after luminal addition of powdered UTP (∼200 μM) suspended in PFC. (B) Plots of ASL height over time following addition of UTP. Normals with UTP alone (♦) or with UTP and bumetanide (10−4 M) (■); CF with UTP alone (▴). N = 11, 6, and 8, respectively. UTP induced a significantly larger increase in ASL height in CF than normal (CF Δ ASL height, 8.4 ± 1.7 μm; normal Δ ASL height, 3.6 ± 0.76 μm; p < 0.05). (C) ASL [Cl−] measured before and after UTP with double-barreled microelectrodes (NL ♦; CF ▴). N = 7 (normals) and 6 (CF) Molecular Cell 2001 8, 149-158DOI: (10.1016/S1097-2765(01)00286-6)

Figure 6 Effect of Osmolyte and Drug Additions on Rotational Mucus Transport in Volume Depleted CF Airway Cultures (A) Mucus solid contents in normal and CF cultures 0, 24, and 72 hr after adding 30 μl PBS to the ASL. All n = 6. * p < 0.05 different from t = 0. † p < 0.01 different from normals. (B) Prolonged exposure (5 s) fluorescence photomicrograph depicting stationary mucus 24 hr after addition of 30 μl PBS to the mucosal surface of a CF culture (left; t = 24 hr); right: image taken 10 min following addition of dry powdered raffinose (8.0 mg) to the mucosal surface (t = 24 hr + 10 min raffinose). (C) Summary data of mucus velocity in CF cultures that had lost rotational mucus transport. Left, mucus velocity 24 hr after volume administration and the same cultures at 24 hr plus 10 min post-UTP addition (open bars; n = 4) or raffinose (filled bars; n = 4); and right, mucus velocity 72 hr after volume administration and the same cultures at 72 hr plus 10 min post-UTP addition (open bars; n = 6) or raffinose (filled bars; n = 6). * indicates mucus velocity faster after UTP and raffinose addition than pre values (p < 0.05). Note that the rotational mucus velocity at 24 hr and 72 hr prior to addition of raffinose and UTP was always 0 Molecular Cell 2001 8, 149-158DOI: (10.1016/S1097-2765(01)00286-6)