Introduction 1 12 1 5 15 16 2+ 17 30 2+ 2+ 2+ 17 30 1 5 31 5 + orpk 32 32 32 2+ 2+ 33 33 2+ Tg737 orpk 20 2+ 17 30 2+ 17 30 2+ 2+ 33 20 Materials and methods Cell culture orpk orpk Tg737 L 32 Tg737 32 Bioluminescence detection of ATP released from epithelial monolayers 34 34 37 34 37 34 37 Fura-2/AM imaging of cytosolic free calcium in a cell monolayer-based perfusion system 38 40 2+ 2+ 2+ 2+ 2+ Materials 34 37 Results Basal ATP release or secretion is not different between mutant and rescued CCD PC monolayers 1 Fig. 1 orpk n left n center right 32 left −10 −9 −8 −7 −6 −5 −4 −3 left middle right  Ionomycin-stimulated ATP release is more robust in rescued versus mutant cell monolayers 2+ 2+ 17 30 2+ 17 30 2+ 17 30 6 8 37 2+ 2 2 2+ 2+ Fig. 2 orpk left left n asterisk P t cross P  Hypotonicity-induced ATP release is more robust in rescued versus mutant cell monolayers 41 44 41 46 + − 41 46 3 7 2+ Fig. 3 orpk left right n 2  Mechanically-induced ATP release is more robust in rescued versus mutant cell monolayers 4 4 2+ Fig. 4 orpk a b 5 2  5 6 7 8 1 2 3 4 2+ Fig. 5 green closed solid balls white open balls  Fig. 6 2+  Fig. 7 Typical real-time course of hypotonic challenge-induced ATP release across the apical cell surface in mutant versus rescued cell monolayers. Hypotonicity induced a marked increase in ATP release that was fivefold more robust in rescued cell monolayers versus mutant cell monolayers. Of interest, however, mechanical stimulation via repeated pipetting near the center of the cell monolayers had no effect on ATP release after hypotonic cell swelling. These data suggest that both stimuli are indeed mechanical in nature and that the same pool of “releasable” ATP is being affected by each stimulus. The slight drop in signal with the pipetting stimulus is the addition of 50 μl of isotonic medium to the 400 μl of 50% diluted medium and the resultant change in osmotic strength at the apical cell surface. The hypotonicity-induced signal observed from mutant cell monolayers is again greatly attenuated  Fig. 8 Typical real-time course of all three stimuli given in an order where each stimulation can be observed in mutant versus rescued cell monolayers. Although the large scale diminished the mechanically induced signal and data, an ATP release transient is observed in rescued cell monolayers and not in mutant cell monolayers. The monophasic ionomycin response is then observed, again more robust in rescued cell monolayers. Then, hypotonic challenge is performed in the presence of ionomycin. Here, the largest values of secreted ATP are observed that approach ∼15–20 μM in the apical medium bathing cilium-competent cell monolayers. In these plots, the effects of the broad specificity anion transport inhibitor, DIDS, are shown. DIDS diminishes ATP release in both types of monolayers and in other epithelial cell monolayer preparations to different degrees. The inhibition is partial and DIDS does not affect the detection reagent. Hexokinase is added at the end of each experiment to eliminate secreted ATP and to diminish the luminescence signal to low levels  5 6 7 8 2+ Flow-induced calcium signals are attenuated in cilium-deficient mutant monolayers versus cilium-competent monolayers 2+ 17 19 2+ 20 30 32 34 40 9 2+ 2+ 2+ 2+ 39 40 47 2+ + 2+ 2+ 39 40 47 2+ 2+ 39 40 47 + 2+ 20 21 29 Fig. 9 2+ 2+ + 2+ 2+ + 2+  10 20 38 2+ 2+ 10 38 2+ 2+ 10 2+ 2+ 2+ 2+ 2+ 2+ 38 2+ 2+ 2+ 2+ 2+ Fig. 10 2+ a 9 b 2+ 2+ 2+ 2+ asterisks a b P t crosses 2+ c asterisks P t 2+ 2+ 2+ 2+  2+ 2+ 2+ 10 2+ 2+ 10 9 2+ 9 2+ 2+ 10 2+ 2+ 10 2+ 30 38 10 2+ + 2+ 2+ 2+ 2+ 17 30 2+ 33 48 54 2+ 33 2+ 10 2+ 2+ + 2+ 2+ 2+ Discussion 11 4 55 2+ 55 4 2+ 56 58 4 59 61 4 5 6 35 36 37 Fig. 11 2+  1 5 62 63 64 67 2+ + + − 17 30 64 67 48 54 68 2+ 2+ 68 2+ + Tg737 orpk 32 69 74 2+ 6 20 33 2+