PMC

Cells were pretreated with either 5 μM of calyculin A, an inhibitor of both protein phosphatase1 (PP1) and protein phosphatase 2A (PP2A) or 5 μM of okadaic acid, a selective PP2A inhibitor and then exposed to 1.0 μM of emetine for 24 hours. RT-PCR was carried out. The results suggest that PP1 not PP2A mediates the effects of emetine on the alternative splicing of Bcl-x. A. Effects of calyculin A in C33A cells. B. Effects of calyculin A in PC3 cells. All experiments were repeated at least three times. C. Effects of calyculin A or okadaic acid on Bcl-x splicing mediated by emetine were quantified and plotted. Ratios of Bcl-xL/Bcl-xS in C33A cells: no treatment, 12.0; with emetine, 4.5; with okadaic acid, 12.0; with emetine and okadaic acid, 5.8; with calyculin A, 12.0; with emetine and calyculin A, 11.8. Ratios of Bcl-xL/Bcl-xS in PC3 cells: no treatment, 7.9; with emetine, 3.0; with okadaic acid, 7.0; with emetine and okadaic acid, 3.1; with calyculin A, 7.0; with emetine and calyculin A, 7.0. +: With emetine, okadaic acid or calyculin A. 0: Without emetine, okadaic acid or calyculin A.

Bath application of calyculin A (20 nM), an inhibitor of PP1, blocked the reductions in spike frequency produced by repeated LSs in Paired cells, when measured as a percent change in spike frequency. (A) Representative light responses recorded from a Paired Type B cell (top trace) or a Paired cell incubated in calyculin A (Paired + Calyculin; bottom trace) for 15 min prior to in vitro extinction. The Paired cell showed a significant decline in light-evoked spike frequency (40%) from the 5th to 30th LS, while the Paired + Calyculin cell showed a 21% increase. (B) Summary spike frequency data of Type B cells exposed to calyculin A. The large reduction in spike frequency observed in Paired (no drug) cells during LSs 16–30 was qualitatively smaller when Paired cells were incubated in calyculin and was no different than the spike frequencies of Untrained cells incubated in calyculin (Unt + Calyculin). (C) Summary data of the relative percent change in B cell spike frequency from the 5th to 30th LS. The incubation of Paired cells in calyculin significantly attenuated the large decrease in spike frequency over the course of 30 LSs normally exhibited by Paired cells without drug. Paired + Calyculin cells spiked at frequencies not statistically different from Unt + Calyculin cells, but frequencies significantly greater than Paired (no drug) cells. Error bars are ± s.e.m. and significant p-values are < 0.05. Note that one cell in the Paired + Calyculin group was omitted from spike frequency analysis during LSs 5–30 and LSs 16–30 because impalement was lost after LS 25.

The incubation of Untrained Type B cells in calyculin A (20 nM final bath concentration) for 15 min prevented the reduction in spike frequency produced by injection of catalytically active PP2B (caPP2B) and increased spiking during the first two LSs. (A) Summary data for average light-evoked spike frequencies during the first two LSs. The combination of caPP2B and calyculin in Untrained cells produced higher spike frequencies compared to cells exposed to either caPP2B or calyculin alone. (B) Representative light responses recorded from an Untrained B cell injected with caPP2B (Unt + PP2B group; top trace) and an Untrained cell injected with caPP2B in the presence of calyculin (Unt + PP2B + Calyculin; bottom trace). The Unt + PP2B cell showed a large reduction in spike frequencies over the course of 30 LSs. Calyculin blocked this caPP2B-produced reduction in spike frequency. (C) Summary spike frequency data for Type B cells exposed to caPP2B and calyculin across 30 LSs. Unt + PP2B cells showed significantly lower spike frequencies during LSs 16–30 compared to Unt + Calyculin cells. In contrast, Unt + PP2B + Calyculin cells showed a slight increase (non-significant) in spike frequencies compared to Unt + Calyculin cells, and were significantly elevated relative to Unt + PP2B cells. (D) Summary data of the relative percent change in B cell spike frequencies from the 5th to 30th LS. Unt + PP2B cells exhibited significant decreases in spike frequency from the 5th to 30th LS (~34%), while Unt + PP2B + Calyculin cells showed very little decrease (~5%) over the course of 30 LSs. Note that data for Unt + Calyculin and Unt + PP2B cells shown here have been replotted from Figures , . Error bars are ± s.e.m. and significant p-values (p's < 0.05) are denoted by an asterisk.

A, left, superimposed plots of the voltage dependence of I_Ca in control (▪), after 5 min in 100nm calyculin A (○) and after 5 min in calyculin A and 1μm Ro 31-8220 (•, n= 6, 2). A, right, similar panel with the reverse order of application (n= 8, 4): Control (▪), 1μm Ro 31-8220 (□) and Ro 31-8220 and 100nm calyculin A (•). B, left, voltage dependence of I_Ca in control (▪), after 5 min in 100nm calyculin A (○) and after 5 min in calyculin A and 200nm Gö 6976 (•, n= 5, 2). B, right, similar panel with the reverse order of application (n= 9, 2): Control (▪); 200nm Gö 6976 (□) and Gö 6976 and 100nm calyculin A (•).