Fig. 5.

Expression of CIRL increases the sensitivity of permeabilized chromaffin cells to the secretion-enhancing effects of α-Ltx. A, Monolayer cultures of nontransfected chromaffin cells were labeled with ³H-norepinephrine (³H-NE), rinsed, and incubated with the indicated concentrations of α-Ltx in physiological saline without Ca²⁺ or Mg²⁺ and with 0.2 mm EGTA for 4 min. The toxin was removed, and the cells were permeabilized for 6 min in KGEP buffer containing 20 μm digitonin and 2 mm MgATP, followed by incubation with or without 30 μm Ca²⁺in KGEP with 2 mm MgATP for 16 min. The incubation solution was removed, the cells were lysed with 1% Triton X-100, and the amount of ³H-NE was determined by liquid scintillation spectrometry. B, C, Chromaffin cells were transfected with plasmids for hGH (pXGH5) and either pCDR7 or pCMVneo, as in Figure 2. After 4–6 d the cells were incubated with the indicated concentrations of α-Ltx in PSS without Ca²⁺ or Mg²⁺ and with 0.1 mm EGTA. After 4 min the toxin was removed, and the cells were permeabilized with 20 μm digitonin in KGEP buffer without Ca²⁺ for 4 min, followed by incubation with or without 30 μm Ca²⁺ in KGEP for 15 min. MgATP (2 mm) was included in both incubations. The amounts of hGH (B) and catecholamine (C) released into the medium and the amounts remaining in the cells were determined as described. For each condition (± CIRL), release in the presence of α-Ltx was normalized to release in the absence of toxin. Actual values for Ca²⁺-dependent release of hGH in the absence of α-Ltx were 21.38 ± 0.80% (pCMVneo) and 15.75 ± 1.78% (pCDR7;p < 0.05 vs pCMVneo). Values for Ca²⁺-dependent release of catecholamine were 17.15 ± 0.59% (pCMVneo) and 17.49 ± 0.72% (pCDR7); n = 4 wells/group.