Dobutamine protects lymphocytes against staurosporin-induced apoptosis: investigation of the antioxidative action of the dobutamine molecule

Introduction

Catecholamines have been shown to modulate various immunological functions. In previous experiments we demonstrated that dobutamine pretreatment protects T cells from staurosporin-induced apoptosis [1]. In the current study we planned to investigate whether antioxidative properties of the dobutamine molecule might be responsible for its protective effect.

Methods

Jurkat T-cell passages 1–12 were used.

Results

Experiments with a caspase-activity assay confirmed previous results: pretreatment (4 hours) with dobutamine 0.1 mM and 0.5 mM decreased staurosporin-induced apoptosis in Jurkat T cells from 14.0% to 11.6% and 8.7%, respectively (P < 0.01). Other catecholamines such as epinephrine and norepinephrine had no protective effect. To investigate whether production of ROS could be measured, Jurkat T cells were loaded with CM-M, H₂DCFDA. After washing steps, the cells were exposed to 0 μ, 1 μM, 10 μM and 100 μM H₂O₂ for 6 hours. The fluorescence signal (ex 480/em 520 nm) measured was 36.7 U, 37.7 U, 38.1 U and 54.3 U, respectively, demonstrating the relation between ROS and the fluorescence signal. Next, production of ROS due to staurosporin treatment (2 μM) was measured: ROS production increased minimally after exposure for 2 hours. Only after 6 hours of staurosporin treatment, the ROS signal increased from 36.7 U to 42.1 U (P < 0.05). Subsequently, the ROS-scavenging effect of dobutamine was investigated. CM-H₂DCFDA-loaded cells were exposed to staurosporin (2 μM) for 2 hours, with or without dobutamine pretreatment (0.1 mM and 0.5 mM): the ROS-scavenging effect was very pronounced in the 0.1 mM group (decrease in fluorescence signal from 56.1 U to 22.5 U, P < 0.01), and increased further in the 0.5 mM group (17.8 U, P < 0.01). Control experiments with unstained cells showed that addition of dobutamine did not change the autofluorescence signal.

Conclusion

These experiments demonstrate that dobutamine acts as a ROS scavenger. Whether this scavenging effect is responsible for the protective properties of dobutamine against staurosporin-induced apoptosis is currently under investigation.