Figure 1.

Loss of Bcl-2 Protein Is Associated with Increased Na⁺-Independent Ca²⁺ Extrusion across the Plasma Membrane

(A) Typical [Ca²⁺]_i trace recorded in a normal (WT) pancreatic acinar cell. Changes in [Ca²⁺]_i were evoked first by application of thapsigargin (Tg) in the absence of external Ca²⁺ and thereafter by exposure, for a period of 400 s, to an external solution containing 10 mM Ca²⁺. The reduction in the elevated [Ca²⁺]_i following removal of the high Ca²⁺ external solution can, in the continued presence of Tg, only be due to Ca²⁺ extrusion across the plasma membrane.

(B) Pancreatic acinar cell from Bcl-2 KO mouse. The same protocol was used as in (A). The rate of reducing [Ca²⁺]_i (due to Ca²⁺ extrusion) after removal of 10 mM external Ca²⁺ was much faster than in the WT cell (shown in A). The resting [Ca²⁺]_i was also lower than in the WT cell.

(C) Comparison of the initial (resting, baseline) [Ca²⁺]_i in WT (blue bar, n = 34) and Bcl-2 KO (red bar, n = 109) pancreatic acinar cells (p < 0.0001). Data in (C) and (E)–(G) are presented as mean ± SEM.

(D) Dependence of the initial rate of Ca²⁺ extrusion on [Ca²⁺]_i, calculated from experiments of the type shown in (A) and (B), i.e., WT (blue, n = 34) and Bcl-2 KO (red, n = 109) pancreatic acinar cells. In cells from Bcl-2 KO mice, Ca²⁺ extrusion was much faster. See also Figure S1.

(E) Bar chart comparing half-times (τ_1/2) of the reduction in [Ca²⁺]_i toward the resting level following removal of external Ca²⁺ in WT (blue bar, n = 20) and Bcl-2 KO (red bar, n = 38) cells.

(F) Bar chart comparing half-times (τ_1/2) of the reduction in [Ca²⁺]_i toward the resting level following removal of external Ca²⁺ in WT pancreatic acinar cells in the normal presence of external Na⁺ (blue bar, n = 18) with those obtained when external Na⁺ was replaced by NMDG⁺ (green bar, n = 24) as well as in Bcl-2 KO cells (red bar, in the presence of Na⁺, n = 6; purple bar, when Na⁺ was replaced by NMDG⁺, n = 25). See also Figure S2.

(G) Bar chart comparing the half-times (τ_1/2) of the reduction in [Ca²⁺]_i toward the resting level following removal of external Ca²⁺ in control AR42J cells (blue bar, n = 14) and in AR42J cells transfected with pcDNA3 Bcl-2 plasmid (Bcl-2 overexpression [oe]) (purple bar, n = 17). Data were collected from cells expressing cytosolic Cameleon YC3.60. See also Figure S3.