1. Aims To examine branching morphogenesis and translumenal potential difference following pharmacological abrogation of chloride secretion. ; To examine whether branching morphogenesis and lumenal chloride secretion are coupled. Methods Whole lung explant cultures E12.5 lungs were explanted from C57/Bl6 mice and placed in chemically-defined serum-free conditions for 48 hours. 30mM Bumetanide was added to the serum-free DMEM/F12, and [Ca 2+ ] o was altered by addition of 1M CaCl 2 to achieve 1.7mM [Ca 2+ ] o concentrations, or omission of 1M CaCl 2 to achieve 1.05mM [Ca 2+ ] o. Trans-lumenal Potential Difference After a 48 hour period in culture 5 MΩ glass electrodes were pushed into a single lumen terminal in each lung. After entry to the lumen had been achieved a five minute equilibration period occurred, before the electrode was removed and the potential difference measured. Determining surface area of lung components Photos of lungs taken at t=0 and t=48 were opened in the programme Adobe Photoshop 7.0. The “lasso” tool was employed to trace around the whole lung or lining of the lung lumen. This area was determined in pixels by Photoshop and change in whole lung, lumen and mesenchymal surface area over 48 hours was calculated (Fig 1 and 2). Conclusions References Introduction The adult lung is an organ for gas exchange. However, in the developing fetus it is filled with fluid. Active chloride secretion, leading to passive electrodiffusion of sodium and osmosis of water, acts as the basis of fluid secretion in the lung [1]. An influx of chloride into the secretory cells of the lung epithelium occurs via the basolateral sodium- potassium-2-chloride cotransporter NKCC1 [2]. Secretion into the lumen of the lung then occurs via the apical channels CFTR [3] and CIC-2. The current accepted hypothesis suggests that fluid secretion in the lung leads to lung growth by increased proliferation and branching morphogenesis [4]. However, preliminary data suggest that the two may actually be uncoupled [5]. This data showed that under high fetal calcium levels branching was suppressed and chloride secretion was enhanced, whilst in low adult calcium levels the opposite is true. By measuring the effect of pharmaceutical inhibition of NKCC1 with Bumetanide (thus preventing fluid secretion) on branching morphogenesis we should be able to clarify the opposing data, both under calcium conditions which stimulate and suppress chloride secretion. Figure 1: Changes in the Surface Area of the lung at 1.05 [Ca 2+ ] o and addition of Bumetanide. (A)Representative E12.5 lungs at t=48 (larger images) and t=0 (smaller images) showing the effect on total and lumenal surface area at increasing doses of Bumetanide under 1.05mM [Ca 2+ ] o. Upper panel from top left shows Control conditions, 0.3  M Bumetanide and 3  M Bumetanide. Lower panel shows 30  M Bumetanide and 300  M Bumetanide application. (B) The total lung, lumen and mesenchymal surface area changes over 48 hours at 1.05mM [Ca 2+ ] o with the application of differing amount of Bumetanide. *=p<0.05, **=p<0.01, performed on absolute branch number. Figure 2: Changes in the Surface Area of the lung at 1.70 [Ca 2+ ] o and addition of Bumetanide. (A)Representative E12.5 lungs at t=48 (larger images) and t=0 (smaller images) showing the effect on total and lumenal surface area at increasing doses of Bumetanide under 1.70mM [Ca 2+ ] o. Upper panel from top left shows Control conditions, 0.3  M Bumetanide and 3  M Bumetanide. Lower panel shows 30  M Bumetanide and 300  M Bumetanide application. (B)The total lung, lumen and mesenchymal surface area changes over 48 hours at 1.70mM [Ca 2+ ] o with the application of differing amount of Bumetanide. *=p<0.05, **=p<0.01, performed on absolute branch number. [1]Olver R.E and Strang L.B. (1974). Ion Fluxes across the Pulmonary Epithelium and The Secretion of Lung Liquid in the Foetal Lamb. Journal of Physiology 241, 327-357 [2] Gillie D.J. et al (2001). Liquid and Ion transport by fetal airway and lung epithelia of mice deficient in sodium- potassium-2-chloride transporter. American Journal of Respiratory Call and Molecular Biology, 25.1 14-20. [3] Nilius. B. and Droogmans. G. (2003) Amazing chloride channels; an overview. Acta Physiologica Scandinavie, 2003, 117, 119-147 [4] Moessinger A.C. et al. (1990). Role of Lung Fluid Volume in Growth and Maturation of the Fetal Sheep Lung. The American Society for Clinical Investigation Inc. 86, 1270-1277 [5] Finney B.A. et al. (2008). Regulation of Mouse Lung Development by the Extracellular Calcium-Sensing Receptor, CaR. Journal of physiology, 586.24, 6007-6019 [6] Vigne.p et al (1994) Na(+)-K(+)-Cl- cotransporter of brain capillary endothelial cells. Properties and regulation by endothelins, hyperosmolar solutions, calyculin A, and interleukin-1. J. Biol. Chem., Vol 269, Issue 31, 19925- 19930 Figure 3: Effect of Bumetanide on developing lung physiology at 1.05 [Ca 2+ ] o (A) Representative E12.5 lungs at t=48 (larger images) and t=0 (smaller images) showing the effect lumen distension (indicating fluid secretion) and branching morphogenesis at increasing doses of Bumetanide at 1.05mM [Ca 2+ ] o. From left shows Control conditions, 0.3  M Bumetanide and 3  M Bumetanide, 30  M Bumetanide and 300  M Bumetanide application. (B) Effect of 1.05mM [Ca 2+ ] o and increasing concentrations of Bumetanide on recorded translumenal potential differences. 30  m and 300  m Bumetanide appears to result in a significant decrease in potential difference. *=p<0.05, **=p<0.01, performed on absolute branch number. (C) Effect of 1.05mM [Ca 2+ ] o and increasing concentrations of Bumetanide on branching morphogenesis. The graph shows the change in number of branches over 24hours (left) and 48 hours (right). A significant increase in branching is seen at 300  m over 24hours but both 300  m and 30  m at 48. *=p<0.05, **=p<0.01, performed on absolute branch number at 24 hours and 48 hours. A B Control 0.3  M Figure 4: Effect of Bumetanide on developing lung physiology at 1.70 [Ca 2+ ] o (A) Representative E12.5 lungs at t=48 (larger images) and t=0 (smaller images) showing the effect lumen distension (indicating fluid secretion) and branching morphogenesis at increasing doses of Bumetanide under 1.70mM [Ca 2+ ] o. From left shows Control conditions, 0.3  M Bumetanide and 3  M Bumetanide, 30  M Bumetanide and 300  M Bumetanide application. (B) Effect of 1.70mM [Ca 2+ ] o and increasing concentrations of Bumetanide on recorded translumenal potential differences. 300  M Bumetanide appears to produce a significant decrease in potential difference. *=p<0.05, **=p<0.01, performed on absolute branch number. (C) Effect of 1.70mM [Ca 2+ ] o and increasing concentrations of Bumetanide on branching morphogenesis. The graph shows the change in branch number over 24hours (left) and 48 hours (right). A significant increase in branching is seen at 300  M only. *=p<0.05, **=p<0.01, performed on absolute branch number at 24 hours and 48 hours.  At 1.05 [Ca 2+ ] o the surface area of the lumen is increased at 300  m Bumetanide only, however, negligible increase is seen in total lung and mesenchymal surface area.  At 1.70 [Ca 2+ ] o the surface area of the lumen is increased by 0.3  m and 3  m Bumetanide, total lung surface area is also increased at this concentration.  At 1.05 [Ca 2+ ] o Bumetanide appears to abrogate transluminal potential difference at both 30  m and 300  m, however, no concentration of Bumetanide appears to effect branching morphogenesis.  At 1.70 [Ca2+]o Bumetanide appears to abrogate translumenal potential difference at 30  m and 300  m, however, an effect on branching morphogenesis is only seen at 300  m. Bumetanide has an IC 50 of 1-5  m [6], suggesting that 30  m and 300  m is too high a concentration to be using, and perhaps here 300  m is having a non specific effect on branching morphogenesis.  As a change in translumenal potential difference is recorded at high levels of Bumetanide, with no change in branching morphogenesis, this suggests that the two may in-fact be uncoupled. A B A B A B The effect of Bumetanide on mouse fetal lung development. C C 3M3M 30  M300  M Control 0.3  M3M3M30  M300  M ** * Bethan Monk ** * * * * *