Chloroquine inhibits Ca2+ permeable ion channels-mediated Ca2+ signaling in primary B lymphocytes

Abstract

Background: Chloroquine, a bitter tastant, inhibits Ca²⁺ signaling, resulting in suppression of B cell activation; however, the inhibitory mechanism remains unclear.

Results: In this study, thapsigargin (TG), but not caffeine, induced sustained intracellular Ca²⁺ increases in mouse splenic primary B lymphocytes, which were markedly inhibited by chloroquine. Under Ca²⁺-free conditions, TG elicited transient Ca²⁺ increases, which additionally elevated upon the restoration of 2 mM Ca²⁺. The former were from release of intracellular Ca²⁺ store and the latter from Ca²⁺ influx. TG-induced release was inhibited by 2-APB (an inhibitor of inositol-3-phosphate receptors, IP₃Rs) and chloroquine, and TG-caused influx was inhibited by pyrazole (Pyr3, an inhibitor of transient receptor potential C3 (TRPC3) and stromal interaction molecule (STIM)/Orai channels) and chloroquine. Moreover, chloroquine also blocked Ca²⁺ increases induced by the engagement of B cell receptor (BCR) with anti-IgM.

Conclusions: These results indicate that chloroquine inhibits Ca²⁺ elevations in splenic B cells through inhibiting Ca²⁺ permeable IP₃R and TRPC3 and/or STIM/Orai channels. These findings suggest that chloroquine would be a potent immunosuppressant.

Keywords: B cells; Ca2+; Chloroquine; IP3R; STIM/Orai channels; TRPC3 channels.

Fig. 1

Chloroquine (chloro) blocks increases of Ca²⁺. a TG induced steady increases of Ca²⁺ in splenic primary B lymphocytes, which were blocked by chloro. The bold line represents the average values. b The average Ca²⁺ levels from 178 cells. c Dose-dependent inhibition of chloro on TG-induced Ca²⁺ increases. ***p < 0.001. These results indicate that chloro attenuates TG-induced elevations of Ca²⁺

Fig. 2

Chloro inhibits extracellular Ca²⁺ influx. a TG induced small transient increases of Ca²⁺ under Ca²⁺-free conditions (0 mM Ca²⁺ and 0.5 mM EGTA). Following the restoration of 2 mM Ca²⁺, large sustained elevations occurred and were declined by chloro. b The average Ca²⁺ levels from 155 cells. ***p < 0.001. These results indicate that chloro inhibits extracellular Ca²⁺ influx

Fig. 3

Chloro blocks TG-induced Ca²⁺ release by inhibiting IP₃Rs on the ER membrane. a After cells were incubated with chloro, neither TG nor the addition of 2 mM Ca²⁺ induced increases of Ca²⁺. These experiments were performed in 241 cells. These data suggest that chloro inhibits TG-induced Ca²⁺ release. b Following cells were incubated with 2-APB, an IP₃R blocker, TG failed to induce increases of Ca²⁺ under Ca²⁺-free conditions (0 mM Ca²⁺ and 0.5 mM EGTA). These experiments were conducted in 129 cells. These data suggest that 2-APB inhibits TG-induced Ca²⁺ release from intracellular Ca²⁺ stores by inhibiting IP₃Rs on the ER membrane

Fig. 4

Caffeine fails to trigger increases of Ca²⁺. Caffeine did not trigger increases of Ca²⁺ under 2 mM Ca²⁺ conditions. These were observed in 227 cells. These results suggest that these cells do not have functional RyRs

Fig. 5

Pyr3 attenuates TG-induced Ca²⁺ elevations. a TG-induced sustained increases of Ca²⁺ were reduced by Pyr3, a selective inhibitor of TRPC3 and STIM/Orai channels. b The average values from 145 cells. ***p < 0.001. These results suggest that TRPC3 and/or STIM/Orai channel mediate TG-induced Ca²⁺ increases

Fig. 6

Chloro inhibits anti-IgM-induced Ca²⁺ increases. a Anti-IgM induced Ca²⁺ increases. b Chloro failed to affect the level of Ca²⁺, however, inhibited anti-IgM-induced increases were inhibited by chloro. c The summary results. NS: p > 0.05; ***p < 0.001. These results indicate that chloro blocks BCR engagement-induced Ca²⁺ increases