The authors have declared that no competing interests exist.
Conceived and designed the experiments: DL ZM. Performed the experiments: ZM ZF ML. Analyzed the data: YC YZ. Contributed reagents/materials/analysis tools: CY YZ. Wrote the paper: DL ZM.
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.
Glibenclamide is widely used to treat type 2 diabetes
Previous studies have found that Ca2+ plays a critical role in the biochemical cascade of signal transduction pathways, resulting in the activation of immune cells
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
Additionally, previous studies found that there was cross-talk between [Ca2+]i and intracellular reactive oxygen species ([ROS]i) signaling generated from mitochondria
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 µg/ml streptomycin and 100 U/ml penicillin at 37°C and in 5% CO2 and 95% air.
Calcium imaging was performed as we described previously
The production of intracellular ROS was monitored by dichlorodihydrofluorescein diacetate (H2DCFDA) as a fluorescent dye. The cells were trypsinized, and the cell suspension was treated with H2DCFDA at a final concentration of 10 µM in the recording solution for 30 min at 37°C. H2DCFDA 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.
The mitochondrial membrane potential was monitored using rhodamine-123 (Rh-123) fluorescent dye imaging. A Raw 264.7 cell suspension was loaded with 10 µg/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.
The standard BS comprised the following (in mM): 130 NaCl, 10 CsCl, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES and 10 glucose titrated to pH 7.4 with NaOH. The BS was then adjusted to an osmotic pressure of 290 mOsm with D-mannitol. The recording BS lacked Ca2+. When indicated, 1.5 mM CaCl2 was added back to the BS. Stock solutions of glibenclamide, pinacidil, diazoxide, diphenyleneiodonium (DPI), H2DCFDA, and Rh-123 were prepared in dimethylsulfoxide (DMSO). 5-hydroxydecanoate (5-HD), tiron, 4-aminopyridine (4-AP), and tetraethylammonium (TEA) were dissolved in distilled H2O. All chemicals were obtained from Sigma (St. Louis, MO, USA) and diluted on the day of the experiment.
The data were expressed as means ± standard errors. The unpaired Student's
Glibenclamide (100 µM) had no effect on the resting [Ca2+]i of Raw 264.7 cells with or without Ca2+ in extracellular solution (
A, representative calcium imaging of Raw 264.7 cells in control solution. B, representative calcium imaging with glibenclamide treatment (100 µM). C, glibenclamide has no effect on the resting [Ca2+]i with or without calcium in the extracellular solution. D, pinacidil and other unselective potassium channel blockers (4-aminopyridine, 4-AP, 10 µM; tetraethylammonium, TEA, 100 µM) did not change the resting [Ca2+]i.
Extracellular ATP (100 µM) induced [Ca2+]i transient elevation in Raw 264.7 cells (
A, representative calcium imaging in control solution. B, calcium imaging with ATP (100 µM), the red arrow showed the peak [Ca2+]i. C, calcium imaging with thapsigargin (Tg, 1 µM), the red arrow showed the peak [Ca2+]i. D, time series of the ratio of fluorescence intensity at excitation wavelengths of 340 and 380 nm (F ratio) during the application of ATP. E, time series of the mean F ratio during the application of Tg. F, time series of the mean F ratio during the sequential application of Tg and ATP. G, 2-aminoethoxydiphenyl borate (2-APB, 100 µM) inhibited the [Ca2+]i transient elevation induced by both ATP and Tg. BS, bath solution.
When Raw 264.7 cells were treated with ATP (100 µM) or Tg (1 µM) in Ca2+-free buffer and then perfused with 1.5 mM Ca2+ extracellular buffer, [Ca2+]i was very modestly increased, which then completely recovered to baseline levels quickly (
Glibenclamide (100 µM) decreased the peak of the [Ca2+]i transient elevation induced by extracellular ATP (100 µM) (
A, C, time series of the mean F ratio during the application of different agents. B, D, the maximal [Ca2+]i with different agents. Glibenclamide (100 µM) and 5-hydroxydecanoate (5-HD, 100 µM) decreased the ATP- or Tg-induced peak [Ca2+]i; pinacidil (100 µM) did not change the maximal [Ca2+]i; diazoxide (Dia, 100 µM) increased the peak [Ca2+]i without significant differences. *
Glibenclamide (100 µM) or 5-HD (100 µM) also decreased the peak of the [Ca2+]i transient elevation induced by extracellular Tg (1 µM) (
The NADPH oxidase (NOX) inhibitor DPI (10 µM) did not decrease the maximal [Ca2+]i induced by extracellular ATP (
A, C, time series of the mean F ratio during the application of different agents. B, D, the maximal [Ca2+]i with different agents. The NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 µM) did not decrease the ATP- or Tg-induced maximal [Ca2+]i. However, the ROS scavenger tiron (1 mM) and mitochondrial inhibitor rotenone (Rot, 5 µM) inhibited the extracellular ATP- and Tg-induced maximal [Ca2+]i. *
Glibenclamide or 5-HD could decrease the level of intracellular ROS detected based on the fluorescence of DCF (
A, representative intracellular ROS imaging in the control solution, the red arrow showed the fluorescence of dichlorofluorescein (DCF). B, representative ROS imaging with glibenclamide (100 µM). C, statistics of intracellular ROS levels with different agents. *
A, representative mitochondrial activity imaging in the control solution, the arrow showed the intensity of rhodamine-123 (Rh-123). B, representative mitochondrial imaging with glibenclamide (100 µM). C, Statistics of the intensity of Rh-123 using different agents. *
When pretreated with tiron and rotenone, glibenclamide and 5-HD could not further decrease the ATP (100 µM)- or Tg (1 µM)-induced maximal [Ca2+]i transient elevation (
When pretreated with tiron and rotenone, glibenclamide could not further decrease the maximal ATP- or Tg-induced [Ca2+]i.
In the present study, for the first time, we have demonstrated the following: 1) glibenclamide cannot affect the resting [Ca2+]i of Raw 264.7 macrophages; 2) extracellular ATP induced the [Ca2+]i transient elevation in Raw 264.7 macrophages by depleting [Ca2+]i stores; 3) intracellular ROS can regulate the ATP-induced [Ca2+]i transient; and 4) glibenclamide decreased the ATP-induced [Ca2+]i transient elevation by inhibiting ROS that were mainly generated from the mitochondria in macrophages.
Cytosolic Ca2+ was considered to be an important second messenger to activate immune cells
At sites of inflammation, macrophages are exposed to various chemical mediators, such as nucleotides, prostanoids, and oxygen radicals
When the calcium stores were pre-released by thapsigargin, ATP could not induce [Ca2+]i transient again. The Ca2+ influx was very modest and recovered to the baseline level quickly when 1.5 mM Ca2+ was reintroduced to the extracellular buffer following pretreatment with ATP or Tg in Ca2+-free buffer. Mikulski Z
Because previous studies showed that glibenclamide has a wide range of anti-inflammatory effects
However, the link between mitochondrial KATP channels and ATP-induced intracellular calcium transient is not clear. Recently, Hänninen SL
First, we used different ROS inhibitors to detect which sources of ROS affected the ATP-induced [Ca2+]i transient elevation. The NOX inhibitor DPI did not decrease the maximal [Ca2+]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 [Ca2+]i. Mitochondria depolarization was previously found to increase the generation of mitochondria-derived ROS, which stimulated Ca2+ sparks in cerebral artery smooth muscle cells
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 KATP channels.
Finally, when pretreated with tiron and rotenone, glibenclamide and 5-HD could not further decrease the maximal ATP- or Tg-induced [Ca2+]i transient. Additionally, glibenclamide had no effect on [Ca2+]i transient in Raw 264.7 macrophages pretreated with 5-HD. Because mitochondrial KATP channels play an important role in ROS production
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 KATP channels, resulting in the inhibition of the cross-talk among [Ca2+]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 [Ca2+]i homeostasis.
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