Korean J Physiol Pharmacol.  2016 Sep;20(5):449-457. 10.4196/kjpp.2016.20.5.449.

N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

• Hasan MA
• Ahn WG
• Song DK

• KMID: 2350501
• DOI: http://doi.org/10.4196/kjpp.2016.20.5.449

Abstract

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca²âº signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca²âº]áµ¢) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca²âº]áµ¢ in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca²âº]áµ¢ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca²âº]áµ¢ in human neutrophils was observed. In Ca²âº-free buffer, NAC- and cysteine-induced [Ca²âº]áµ¢ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca²âº]áµ¢ in human neutrophils occur through Ca²âº influx. NAC- and cysteine-induced [Ca²âº]áµ¢ increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na⁺-free HEPES, both NAC and cysteine induced a marked increase in [Ca²âº]áµ¢ in human neutrophils, arguing against the possibility that Na⁺-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca²âº]áµ¢ increasing activity. Our results show that NAC and cysteine induce [Ca²âº]áµ¢ increase through Ca²âº influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

Keyword

Acetyl-L-cysteine; Cysteine; Human neutrophils; Intracellular free-calcium concentration

MeSH Terms

Acetylcysteine*
Calcium Channels
Calcium*
Cysteine*
HEPES
Humans*
Neutrophils*
Ruthenium
Ruthenium Red
Acetylcysteine
Calcium
Calcium Channels
Cysteine
HEPES
Ruthenium
Ruthenium Red

Figure

• 

  Fig. 1 Concentration-dependent effects of NAC and cysteine on [Ca2+]i in human neutrophils.(A) NAC (100 nM~1 mM) or (C) cysteine (100 nM~1 mM) was treated at 300 s following 5 min pre-read. [Ca2+]i was measured as described in methods and materials. Changes in [Ca2+]i were expressed as the relative fluorescence intensity of Fluo-3 AM over baseline fluorescence intensity (F/F0). (B)&(D) [Ca2+]i following treatment of NAC and cysteine was shown as area under curve (AUC), which was calculated for 900 s (300~1200 s) and was expressed as percentage control (% control). Data were analyzed by Graphpad Prism 5.0 (Graphpad software) using ANOVA. Bonferroni test was used for post-hoc comparison. An average of four (A, B) and three (C, D) independent experiments is shown. ns, no significant difference, **p<0.01, ***p<0.001.

• 

  Fig. 2 Effects of NAC and cysteine on fMLP-induced increase in [Ca2+]i in human neutrophils.Vehicle or NAC at 10 µM, 100 µM and 1 mM (A~C), and fMLP (1 µM) were added at the time points indicated by the arrowheads. Changes in [Ca2+]i were expressed as the relative fluorescence intensity of Fluo-3 AM over baseline fluorescence intensity (F/F0). (D) [Ca2+]i following treatment of vehicle or fMLP (1 µM) was shown as area under curve (AUC), which was calculated for 300 s (900~1200 s) and was expressed as percentage control (% control). Data were analyzed by Graphpad Prism 5.0 (Graphpad software) using ANOVA. Bonferroni test was used for post-hoc comparison. An average of three independent experiments is shown. ***p<0.001.

• 

  Fig. 3 NAC and cysteine had no effect on [Ca2+]i in human neutrophils in Ca2+-free HEPES buffer.(A) NAC (100 nM~1 mM) or (B) cysteine (100 nM~1 mM) was treated at 300 s following 5 min pre-read. [Ca2+]i was measured as described in methods and materials. Changes in [Ca2+]i were expressed as the relative fluorescence intensity of Fluo-3 AM over baseline fluorescence intensity (F/F0).

• 

  Fig. 4 SKF96365 and ruthenium red inhibit NAC-induced [Ca2+]i increase in human neutrophils.Ca2+-channel inhibitors (A) SKF96365 (10 µM) and (C) ruthenium red (20 µM), and NAC (100 µM) were added at the time points indicated by the arrowheads. [Ca2+]i was measured as described in methods and materials. Changes in [Ca2+]i were expressed as the relative fluorescence intensity of Fluo-3 AM over baseline fluorescence intensity (F/F0). (B, D) [Ca2+]i following addition of Ca2+-channel inhibitors were shown as area under curve (AUC), which was calculated for 300 s (900~1200 s) and was expressed as percentage control (% control). Data were analyzed by Graphpad Prism 5.0 (Graphpad software) using ANOVA. Bonferroni test was used for post-hoc comparison. An average of three independent experiments is shown. ns, no significant difference, **p<0.01.

• 

  Fig. 5 SKF96365 and ruthenium red inhibit cysteine-induced [Ca2+]i increase in human neutrophils.Ca2+-channel inhibitors (A) SKF96365 (10 µM) and (C) ruthenium red (20 µM), and cysteine (10 µM) were added at the time points indicated by the arrowheads. [Ca2+]i was measured as described in methods and materials. Changes in [Ca2+]i were expressed as the relative fluorescence intensity of Fluo-3 AM over baseline fluorescence intensity (F/F0). (B, D) [Ca2+]i following addition of Ca2+ channel inhibitors were indicated as area under curve (AUC), which was calculated for 300 s (900~1200 s) and was expressed as percentage control (% control). Data were analyzed by Graphpad Prism 5.0 (Graphpad software) using ANOVA. Bonferroni test was used for post-hoc comparison. An average of three independent experiments is shown. ns, no significant difference, *p<0.05.

• 

  Fig. 6 NAC and cysteine increase [Ca2+]i in human neutrophils in Na+-free HEPES buffer.(A) NAC (100 nM~1 mM) or (C) cysteine (100 nM~1 mM) was treated at 300 s following 5 min preread. [Ca2+]i was measured as described in methods and materials. Changes in [Ca2+]i were expressed as the relative fluorescence intensity of Fluo-3 AM over baseline fluorescence intensity (F/F0). (B)&(D) [Ca2+]i following treatment of NAC and cysteine was shown as area under curve (AUC), which was calculated for 900 s (300~1200 s) and was expressed as percentage control (% control). Data were analyzed by Graphpad Prism 5.0 (Graphpad software) using ANOVA. Bonferroni test was used for post-hoc comparison. An average of three independent experiments is shown. ns, no significant difference, ***p<0.001.

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