Congo red modulates ACh-induced Ca(2+) oscillations in single pancreatic acinar cells of mice

Abstract

Aim: Congo red, a secondary diazo dye, is usually used as an indicator for the presence of amyloid fibrils. Recent studies show that congo red exerts neuroprotective effects in a variety of models of neurodegenerative diseases. However, its pharmacological profile remains unknown. In this study, we investigated the effects of congo red on ACh-induced Ca(2+) oscillations in mouse pancreatic acinar cells in vitro.

Methods: Acutely dissociated pancreatic acinar cells of mice were prepared. A U-tube drug application system was used to deliver drugs into the bath. Intracellular Ca(2+) oscillations were monitored by whole-cell recording of Ca(2+)-activated Cl(-) currents and by using confocal Ca(2+) imaging. For intracellular drug application, the drug was added in pipette solution and diffused into cell after the whole-cell configuration was established.

Results: Bath application of ACh (10 nmol/L) induced typical Ca(2+) oscillations in dissociated pancreatic acinar cells. Addition of congo red (1, 10, 100 μmol/L) dose-dependently enhanced Ach-induced Ca(2+) oscillations, but congo red alone did not induce any detectable response. Furthermore, this enhancement depended on the concentrations of ACh: congo red markedly enhanced the Ca(2+) oscillations induced by ACh (10-30 nmol/L), but did not alter the Ca(2+) oscillations induced by ACh (100-10000 nmol/L). Congo red also enhanced the Ca(2+) oscillations induced by bath application of IP3 (30 μmol/L). Intracellular application of congo red failed to alter ACh-induced Ca(2+) oscillations.

Conclusion: Congo red significantly modulates intracellular Ca(2+) signaling in pancreatic acinar cells, and this pharmacological effect should be fully considered when developing congo red as a novel therapeutic drug.

Figure 1

Chemical structure of congo red.

Figure 2

Congo red potentiates ACh-induced Ca²⁺ oscillations in a concentration-dependent manner. (A–C) Representative traces showing that in the presence of 10 nmol/L ACh, bath-applied congo red enhanced Ca²⁺ oscillations in a concentration-dependent manner. Traces (A–C) were recorded from the same cell. (D) The bar graph indicates potentiation of the net charge of the normalized current in the ACh-induced response by different concentrations of congo red. In this and the following figures, the numbers inside each column indicate the number of cells tested. The vertical bars represent the Mean±SEM. ^cP<0.01.

Figure 3

Congo red alone fails to induce a detectable current response under patch-clamp recording conditions. (A–C) Bath-applied congo red alone at different concentrations failed to induce any detectable current response. However, when ACh (10 nmol/L) was immediately bath-applied after congo red application, it induced enhanced Ca²⁺ oscillations in a concentration-dependent manner. (D) The bar graph summarizes the effects of ACh-induced current responses (normalized net charge) with different concentrations of congo red pretreatment. ^cP<0.01. These results suggest that congo red itself is not able to induce Ca²⁺ oscillations, but it clearly potentiates ACh-induced Ca²⁺ oscillations.

Figure 4

Effects of congo red on different concentrations of ACh-induced Ca²⁺ oscillations. (A) Representative traces of patch-clamp, whole-cell recordings, in which congo red (30 μmol/L) potentiated 30 nmol/L ACh-induced Ca²⁺ oscillations (Aa), but not 100 nmol/L ACh-induced Ca²⁺ responses (Ab). The trace shown represents the similar effects of congo red observed in the 5 other cells tested. (B) Representative traces of Ca²⁺ imaging, in which congo red (30 μmol/L potentiated 30 nmol/L ACh-induced Ca²⁺ oscillations (Ba), but not 100 nmol/L ACh-induced Ca²⁺ responses (Bb). A similar effect of congo red to that shown in Ba was observed in 66 cells (from 5 mice), and similar effects to those shown in Bb were observed in 44 cells (from 3 mice).

Figure 5

Effects of congo red on the Ca²⁺ oscillations induced by intracellular application of IP₃. (Aa) A typical trace showing that when the recording electrode contained 30 μmol/L IP₃, the formation of a whole-cell recording by suction of the recording electrode (indicated by an arrow) induced pulsatile Ca²⁺ oscillations. (Ab) Under conditions of intracellular IP₃-induced Ca²⁺ oscillations, bath-application of 10 μmol/L congo red enhanced the IP₃ response. (B) The bar graph summarizes the data regarding IP₃-induced Ca²⁺ oscillations before, during and after washout of congo red. ^bP<0.05, ^cP<0.01.

Figure 6

Effects of intracellular congo red on ACh-induced Ca²⁺ oscillations. (A) A representative trace showing the patch recording mode switch from perforated patch to whole-cell recording (without congo red in the pipette solution) in the presence of 10 nmol/L bath ACh. (B) The same recording mode switch with a recording electrode containing 10 μmol/L congo red (a), followed by bath application of 10 μmol/L congo red (b). (C) A summary of pooled data from 6A and 6B showing that, in contrast to extracellular application, intracellular application of congo red did not affect ACh-induced Ca²⁺ oscillations. Therefore, congo red potentiates ACh-induced Ca²⁺ oscillations via an extracellular target.

Figure 7

Effects of 2-APB, a well-known SOCC blocker, on ACh-induced Ca²⁺ oscillations. (A) A representative typical trace showing that application of 30 μmol/L congo red enhanced ACh (30 nmol/L)-induced Ca²⁺ oscillations. (B) A representative typical trace showing that application of 30 μmol/L 2-APB enhanced ACh-induced Ca²⁺ oscillations. Both congo red and 2-APB potentiated ACh-induced Ca²⁺ oscillations in the same manner, which suggests that congo red may block SOCCs in pancreatic acinar cells.