FIGURE 5.

ABA induces an increase of the [Ca²⁺]_i and of the [cAMP]_i in HeLa co-transfected with CD38 and LANCL2 through a G_i-dependent mechanism. 24 h after transfection with the empty vector (gray traces) or with LANCL2 plasmid (black traces), CD38⁺ (A) or CD38⁻ (B) HeLa were loaded with Fura2/AM and exposed to the indicated ABA concentrations at 20 °C. The [Ca²⁺]_i was measured as described before (16). Each microscopic field contained ≥20 cells. Representative traces from three experiments, yielding closely comparable results, are shown. Inset: Western blot analysis of cell lysates from control (CD38⁺) and CD38⁺/LANCL2⁺ HeLa stained with a monoclonal antibody against the V5 epitope. C, CD38⁺ HeLa transfected with LANCL2 (CD38⁺/LANCL2⁺) were preincubated without (triangles), or with (squares) 2 μg/ml PTX for 1 h, then challenged with 100 nm ABA and processed for measurement of [cAMP]_i levels. Alternatively, CD38⁺/LANCL2⁺ HeLa were further transfected with α_t (circles) and then challenged with 100 nm ABA. No increase of the [cAMP]_i was observed upon addition of ABA in the following controls: CD38⁺ HeLa transfected with the empty vector for LANCL2; CD38⁺ HeLa transfected with α_t alone (in the absence of LANCL2) (not shown). *, p < 0.01 by t test, compared with values measured in cells transfected with the empty vector, or with α_t, or compared with values measured in PTX-preincubated cells. D, CD38⁺ HeLa were transfected with LANCL2-pcDNA6.2/V5/GW/D-TOPO^© and without, control or with α_t (pCS2+/α-transducin). After 24 h, cells were loaded with Fura2/AM, preincubated for 1 h at 37 °C without or with 2 μg/ml PTX, and then exposed to 100 nm ABA at 25 °C. The [Ca²⁺]_i was measured as described before (19). Each microscopic field contained ∼20 cells. Representative traces from three experiments, yielding closely comparable results, are shown. E, CD38⁺/LANCL2⁺ HeLa were transfected, or not (triangles), with Gα_q/i (circles) and then challenged with 100 nm ABA and processed for measurement of [IP₃]_i levels. No significant increase of the [IP₃]_i was observed upon addition of ABA to CD38⁺ HeLa transfected with Gα_q/i alone (in the absence of LANCL2, squares). *, p < 0.05 by t test, compared with values measured in cells not transfected with Gα_q/i. Results in C and D are expressed as percentage increase over basal values, measured at time = 0 for each cell type (n = 4).