Effect of prostaglandin E₂ on ATP-induced Ca²⁺ responses in human THP-1 monocytic cells

Introduction

To clarify the relation between ATP and prostaglandin E₂ (PGE₂) in the immunologic system, we investigated the acute and chronic effects of PGE₂ on activation of purinergic signaling in monocytes by measuring the ATP-induced elevation of intracellular Ca²⁺([Ca]_i) in fura-2-loaded THP-1 monocytes.

Method

THP-1 monocytes were grown for about 2 days. To examine the chronic effects, PGE₂ and dibutyryl cAMP (dbcAMP) were added and incubated for another day. The cell suspensions were washed, loaded with fura-2-AM, and transferred into a quartz cuvette and placed in the thermostat-regulated sample chamber of a dual excitation beam spectrophotometer. To examine the acute effects, ATP was added immediately after PGE₂ and dbcAMP into the cuvette. In the chronic experiment, ATP alone was added into the cuvette. Fura-2 fluorescence emission was measured at 510 nm. The [Ca]_i was calculated from the ratio of the fluorescence at the two excitation wavelengths.

Results

ATP induced a transient increase in [Ca]_i followed by a sustained elevation of [Ca]_i. Acutely, PGE₂ inhibited both the transient and sustained ATP-induced elevations of [Ca]_i. However, this acute inhibitory effect diminished gradually with time and chronic PGE₂ accelerated the transient and sustained ATP-induced [Ca]_i elevations for 24 hours. Both the acute and chronic effects of PGE₂ were mimicked by dbcAMP. In Ca²⁺-free solution, ATP did not induce the sustained elevation of [Ca]_i in control cells or cells pretreated for 24 hours with dbcAMP. This indicates that the ATP-induced sustained elevation of [Ca]_i was due to Ca²⁺ entry. In addition, receptor-operated Ca²⁺ channel blockers inhibited the sustained ATP-induced elevation of [Ca]_i in control cells and cells pretreated with for 24 hours dbcAMP.

Conclusion

Acute PGE₂ inhibited the ATP-induced activation of monocytes. On the other hand, chronic PGE₂ accelerated monocyte activation by upregulation of receptor-operated Ca²⁺ channels ROCs). If this mechanism exhibits a physiological role, ROC inhibitors should be developed as new anti-inflammatory agents.