Glibenclamide Decreases ATP-Induced Intracellular Calcium Transient Elevation via Inhibiting Reactive Oxygen Species and Mitochondrial Activity in Macrophages

Duo-Ling Li,Zhi-Yong Ma,Chuan-Zhu Yan,Yun Zhang,Ming-Ying Ling,Zhi-Jie Fu,Xiongwen Chen

doi:10.1371/journal.pone.0089083

Abstract

Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP)-induced intracellular calcium ([Ca2+]i) handling in Raw 264.7 macrophages. In the present study, [Ca2+]i transient, reactive oxygen species (ROS) and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K+ channel blockers had no effect on the resting [Ca2+]i of Raw 264.7 cells. Extracellular ATP (100 µM) induced [Ca2+]i transient elevation independent of extracellular Ca2+. The transient elevation was inhibited by an ROS scavenger (tiron) and mitochondria inhibitor (rotenone). Glibenclamide and 5-hydroxydecanoate (5-HD) also decreased ATP-induced [Ca2+]i transient elevation, but pinacidil and other unselective K+ channel blockers had no effect. Glibenclamide also decreased the peak of [Ca2+]i transient induced by extracellular thapsigargin (Tg, 1 µM). Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca2+]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca2+]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.

Highlights

Glibenclamide is widely used to treat type 2 diabetes [1]
We found that glibenclamide could ameliorate the progression of atherosclerosis and reduce the production of inflammatory cytokines as well as the phosphorylation of p65 and ERK1/2 in Raw 264.7 macrophages [5]
When the intracellular calcium stores were released by Tg (1 mM), adenosine triphosphate (ATP) could not induce [Ca2+]i transient elevation again (Figure P,0.05, 2C, F). 2-APB (100 mM) was able to block the [Ca2+]i transient elevation induced by both ATP and Tg (P,0.05, Figure 2F)

Summary

Introduction

Glibenclamide is widely used to treat type 2 diabetes [1]. The pharmacological action of glibenclamide is to inhibit adenosine triphosphate (ATP)-sensitive K+ channels (KATP) in pancreatic b cells, leading to the stimulation of insulin secretion [2]. Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects [3,4]. We found that glibenclamide could ameliorate the progression of atherosclerosis and reduce the production of inflammatory cytokines as well as the phosphorylation of p65 and ERK1/2 in Raw 264.7 macrophages [5]. The mechanism responsible for the anti-inflammatory effect of glibenclamide is largely unexplored. Because glibenclamide was found to increase the intracellular Ca2+ concentration ([Ca2+]i) in pancreatic b cells [2], investigating whether glibenclamide was able to affect [Ca2+]i in Raw 264.7 macrophages was considered worthwhile

Methods

Results

Conclusion