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

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.


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 (K ATP ) in pancreatic b cells, leading to the stimulation of insulin secretion [2]. Meanwhile, increasing evidence has revealed that glibenclamide also has a wide range of anti-inflammatory effects [3,4]. Recently, 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]. However, the mechanism responsible for the anti-inflammatory effect of glibenclamide is largely unexplored.
Previous studies have found that Ca 2+ plays a critical role in the biochemical cascade of signal transduction pathways, resulting in the activation of immune cells [6,7]. Because glibenclamide was found to increase the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in pancreatic b cells [2], investigating whether glibenclamide was able to affect [Ca 2+ ] i in Raw 264.7 macrophages was considered worthwhile.
As the main effector cells at sites of inflammation and tissue injury, macrophages are likely to be exposed to many extracellular molecules that are involved in cellular signaling [8,9]. In particular, extracellular ATP was found to be one of the key molecules in modulating the immune response through their capacity to bind and activate multiple nucleotide receptor family members [10]. In non-excitable cells, extracellular ATP induces an elevation of cytosolic calcium by two distinct mechanisms, either by the activation of Ca 2+ release from intracellular Ca 2+ stores or by the activation of Ca 2+ influx from the extracellular medium [11,12]. However, it is unclear whether glibenclamide has any effect on ATP-induced [Ca 2+ ] i handling.
Additionally, previous studies found that there was cross-talk between [Ca 2+ ] i and intracellular reactive oxygen species ([ROS] i ) signaling generated from mitochondria [13,14]. As we know, glibenclamide can block mitochondrial K ATP channels, which play an important role in [ROS] i production [15]. Therefore, we hypothesized that [ROS]

Cell culture
Murine macrophage cell line Raw 264.7 cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 10% fetal calf serum, 100 mg/ml streptomycin and 100 U/ml penicillin at 37uC and in 5% CO 2 and 95% air.

Intracellular calcium measurements
Calcium imaging was performed as we described previously [16]. Briefly, Raw 264.7 cells were incubated with 2 mM fura-2/ acetoxymethylester for 30 min at 37uC and then were washed out at room temperature for another 30 min. Measurements were made using an inverted microscope (Nikon TE2000-U, Nikon, Japan) and a TILLvisION digital imaging system (TILL Photonics GmbH, Munich, Germany) as reported previously [16]. [Ca 2+ ] i was indicated as the ratio of fluorescence intensity at excitation wavelengths of 340 and 380 nm (F ratio). The emission wavelength was 510 nm. The background intensity was subtracted from the fluorescent intensity changes, and the resulting [Ca 2+ ] i values were normalized as the differences between the fluorescence intensities with different agents and the intensity in standard bath solution (BS) by averaging the values of at least 50 cells/treatment.

Measurement of intracellular ROS levels
The production of intracellular ROS was monitored by dichlorodihydrofluorescein diacetate (H 2 DCFDA) as a fluorescent dye. The cells were trypsinized, and the cell suspension was treated with H 2 DCFDA at a final concentration of 10 mM in the recording solution for 30 min at 37uC. H 2 DCFDA is oxidized to the fluorescent dichlorofluorescein (DCF), which is monitored at excitation and emission wavelengths of 488 and 510 nm, respectively, using a TILLvisION digital imaging system. After incubating cells with different reagents, the ROS levels were determined by comparing the changes in fluorescence intensity with that in the standard extracellular recording solution. The fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment.

Detection of mitochondrial membrane potential
The mitochondrial membrane potential was monitored using rhodamine-123 (Rh-123) fluorescent dye imaging. A Raw 264.7 cell suspension was loaded with 10 mg/ml Rh-123 at room temperature for 15 min. After loading, the cells were continuously perfused with recording solution. Rh-123 fluorescence images were captured using the method described above. The fluorescence was excited at 490 nm and filtered at 530 nm. The fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment.

Data analysis
The data were expressed as means 6 standard errors. The unpaired Student's t-test and one-way ANOVA were used for statistical analysis where appropriate. P,0.05 was considered statistically significant.

Effect of glibenclamide on the resting [Ca 2+ ] i of Raw 264.7 macrophages
Glibenclamide (100 mM) had no effect on the resting [Ca 2+ ] i of Raw 264.7 cells with or without Ca 2+ in extracellular solution (P.0.05, Figure 1A When Raw 264.7 cells were treated with ATP (100 mM) or Tg (1 mM) in Ca 2+ -free buffer and then perfused with 1.5 mM Ca 2+ extracellular buffer, [Ca 2+ ] i was very modestly increased, which then completely recovered to baseline levels quickly ( Figure S2A

Effect of glibenclamide on intracellular ROS and mitochondrial activity
Glibenclamide or 5-HD could decrease the level of intracellular ROS detected based on the fluorescence of DCF (P,0.05, Figure 5). Pinacidil and other unselective potassium channel blockers (TEA and 4-AP) did not change intracellular ROS (P.0.05, Figure 5). Glibenclamide or 5-HD also decreased the fluorescence intensity of Rh-123, reflecting the depolarization of the mitochondrial membrane potential (P,0.05, Figure 6). Diazoxide could increase the intensity of Rh-123 and intracellular ROS (P,0.05, Figure 6). When simultaneously pretreated with glibenclamide (100 mM) and 5-HD (100 mM), the fluorescence intensity of DCF (P.0.05, Figure 5) and Rh-123 (P.0.05, Figure 6) was decreased to the same level as that individually decreased by glibenclamide or 5-HD.  Figure 7). Cytosolic Ca 2+ was considered to be an important second messenger to activate immune cells [6,7]. Resting lymphocytes were found to maintain a low concentration of [Ca 2+ ] i [6]. Cytosolic Ca 2+ elevations are required for diverse immune cellular functions, including differentiation, effector function, and gene transcription [7,17]. Recently, 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 [5]. However, it is unclear that glibenclamide can affect [Ca 2+ ] i in macrophages. In the present study, we found that glibenclamide did not affect the resting [Ca 2+ ] i of Raw 264.7 macrophages, which was very different than the effect of glibenclamide on excitable cells, such as smooth muscle cells [18], neurons [19], and b-cells [2]. In these cells, glibenclamide inhibits membrane K ATP , leading to membrane depolarization, which in turn opens voltage-gated calcium channels and increases [Ca 2+ ] i via Ca 2+ influx.

Discussion
At sites of inflammation, macrophages are exposed to various chemical mediators, such as nucleotides, prostanoids, and oxygen radicals [8,9]. In particular, increasing evidence has suggested that extracellular ATP participates in the inflammatory response as a proinflammatory mediator through its capacity to bind and activate multiple nucleotide receptor family members [10]. In non-excitable cells, extracellular ATP induces an elevation of cytosolic Ca 2+ . In the present study, we found that ATP induced [Ca 2+ ] i transient elevation independent of extracellular Ca 2+ in Raw 264.7 cells. We also found this phenomena in THP-1 cells, whereas other studies found that ATP induced [Ca 2+ ] i independent of extracellular Ca 2+ in Sertoli cells [20], the principal cells of the inner medullary collecting duct [21], and human monocytes [22]. Thus, ATP induced [Ca 2+ ] i transient independent of extracellular Ca 2+ in a manner that was not specific to macrophages.
When the calcium stores were pre-released by thapsigargin, ATP could not induce [Ca 2+ ] i transient again. The Ca 2+ influx was very modest and recovered to the baseline level quickly when 1.5 mM Ca 2+ was reintroduced to the extracellular buffer following pretreatment with ATP or Tg in Ca 2+ -free buffer. Mikulski Z et al [23] also found that nicotinic receptors on rat alveolar macrophages dampened the ATP-induced Ca 2+ -release from intracellular stores. In murine J774 macrophages, low concentrations (10 mM) of ATP evoked Ca 2+ transient in a phospholipase C (PLC)-dependent manner [24]. These results suggested that the Ca 2+ influx resulting from the depletion of Ca 2+ stores was modest and transient in Raw 264.7 cells and was covered by ATP-or Tg-induced [Ca 2+ ] i release. Furthermore, we found that 2-APB, a membrane permeable IP 3 receptor antagonist [25], was able to inhibit the [Ca 2+ ] i elevation induced by ATP. Many studies found that ATP induced Ca 2+ release from the endoplasmic reticulum (ER) through inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) [26,27], and these results suggested that ATP induced the [Ca 2+ ] i transient elevation by releasing Ca 2+ from the intracellular Ca 2+ stores in Raw 264.7 macrophages.
Because previous studies showed that glibenclamide has a wide range of anti-inflammatory effects [3,4] Because glibenclamide has been shown to have an inhibitory effect on mitochondria K ATP channels [28], the results suggested that However, the link between mitochondrial K ATP channels and ATP-induced intracellular calcium transient is not clear. Recently, Hä nninen SL et al [29] found that mitochondrial uncoupling downregulated calsequestrin expression and reduced sarcoplasmic reticulum Ca 2+ stores in cardiomyocytes. Thus, we hypothesized that ROS mainly from the mitochondria participated in the regulation of ATP-induced [Ca 2+ ] i transient and that glibenclamide might inhibit this transient by inhibiting ROS generation by blocking mitochondrial K ATP channels.
First, we used different ROS inhibitors to detect which sources of ROS affected the ATP-induced [Ca 2+ ] i transient elevation. The NOX inhibitor DPI did not decrease the maximal [Ca 2+ ] i transient induced by extracellular ATP or Tg. An ROS scavenger (tiron) and a mitochondrial inhibitor (rotenone) inhibited the extracellular ATP-or Tg-induced maximal [Ca 2+ ] i . Mitochondria depolarization was previously found to increase the generation of mitochondria-derived ROS, which stimulated Ca 2+ sparks in cerebral artery smooth muscle cells [30]. Rakesh Rathorea et al [31] found that hypoxia might specifically increase mitochondrial ROS generation, which subsequently contributed to a hypoxiainduced increase in [Ca 2+ ] i and contraction in the pulmonary artery smooth muscle cells. These results suggested that ROS mainly from the mitochondria participated in the regulation of the ATP-induced intracellular calcium in Raw 264.7 macrophages.
Second, we explored whether glibenclamide could decrease the level of intracellular ROS. Glibenclamide or 5-HD could decrease the level of intracellular ROS, but pinacidil and other unselective potassium channel blockers (TEA and 4-AP) did not change the intracellular ROS. Glibenclamide and 5-HD also decreased the intensity of Rh-123 staining, reflecting depolarization of the mitochondrial membrane potential. Diazoxide was able to increase the intensity of Rh-123 staining and the intracellular ROS level. Glibenclamide and 5-HD showed no additive effect in decreasing the fluorescence intensity of DCF and Rh-123. Thus, glibenclamide could be concluded to decrease the level of intracellular ROS by inhibiting mitochondrial activity and blocking mitochondrial K ATP channels.
Finally, when pretreated with tiron and rotenone, glibenclamide and 5-HD could not further decrease the maximal ATP-or Tginduced [Ca 2+ ] i transient. Additionally, glibenclamide had no effect on [Ca 2+ ] i transient in Raw 264.7 macrophages pretreated with 5-HD. Because mitochondrial K ATP channels play an important role in ROS production [15], we could conclude that the inhibitory effect of glibenclamide on the ATP-induced [Ca 2+ ] i transient elevation might be mediated by inhibiting ROS and blocking mitochondrial K ATP channels.
In conclusion, extracellular ATP induced the intracellular calcium transient elevation by depleting calcium stores in Raw 264.7 macrophages, which could be regulated by ROS mainly from mitochondria. Glibenclamide might inhibit this transient activity by blocking mitochondrial K ATP channels, resulting in the inhibition of the cross-talk among [Ca 2+ ] i , intracellular ROS, and mitochondrial activity. However, further studies should be performed to reveal the effect of glibenclamide on other physiological functions of macrophage cells by regulating [Ca 2+ ] i homeostasis.