Figure 2. Effect of deletion of Orai1 on Ca²⁺ signaling, enzymes content and exocytosis.

All experiments are with Orai1^fl/fl mice maintained on solid diet (controls) and Orai1^−/− mice maintained on liquid diet for 10 days after last gavage with Tamoxifen, except in (m).

(a) Ca²⁺ signaling evoked by carbachol stimulation in Orai1^fl/fl (black trace) and Orai1^−/− (red trace) acinar cells in Ca²⁺ containing medium. Where indicated the cells were exposed to 0 and 5 mM external Ca²⁺ to assay Ca²⁺ influx.

(b, c) Acinar cells in Ca²⁺-free solution were stimulated with 100 μM carbachol (b) or 25 μM CPA (c) to assay Ca²⁺ release from stores and then exposed to 5 mM external Ca²⁺ to determine store-operated Ca²⁺ influx. Traces in (a–c) are mean±s.e.m of the number of acini obtained from 4 mice of each line. Each acinus comprised of 5–10 cells.

(d, e) Orai1^fl/fl (d) and Orai1^−/− acinar cells (e) were stimulated with 2 pM CCK8 to evaluate receptor-stimulated Ca²⁺ oscillations.

(f) Summary of Ca²⁺ release and influx. Here and in (g, i, k) the p values are listed in the columns.

(g) Summary of Ca²⁺ oscillation frequency recorded in 64 Orai1^fl/fl and 80 Orai1^−/− acinar cells.

(h) Protein levels of the granule markers syntaxin 3 and VAMP8, and of actin. The columns show the average staining intensity relative to actin and is plotted as mean±s.e.m.

(i–k) Activity of total lipase (i, 6 mice), amylase (j, 9 mice) and trypsin (k, 3 mice) in Orai1^fl/fl (black) and Orai1^−/− acinar cells (red).

(l) Time course of exocytosis stimulated by 100 pM CCK8 in acini obtained from age-matched Orai1^fl/fl and Orai1^−/− mice 10 (blue, circles), 30 (red, triangles) and 112 (green, squares) days after deletion of Orai1.

(m) Chymotrypsin in feces of Orai1^fl/fl mice (black) and Orai1^−/− mice maintained on solid (red) and liquid diets (blue) for 10 days after final Tamoxifen gavage.