Enhanced expression of gene coding for β-endorphin in human monocytic cells exposed to pulsed radio frequency electric fields through thermal and non-thermal effects

Abstract

Background: The enhanced expression of endogenous opioid peptides, including β-endorphin, has been implicated in the mechanism of action of pulsed radio frequency (PRF) application in pain modulation. Because thermal effects cannot be separated from the physical property of PRF application to biological tissues, we evaluated whether temperatures higher than that of the normal body temperature (37°C) modulate mRNA expression for the precursor of β-endorphin, proopiomelanocortin (POMC) in human monocytic cells THP-1. We also attempted to examine whether mechanisms other than thermal effects also modulate such gene expression.

Methods and results: The mRNA for POMC in THP-1 cells increased by a 15-minutes incubation at 42°C, 45°C, or 70°C without PRF application as compared with that in cells incubated at 37°C. On the other hand, gene expression for POMC in cells incubated at 20°C as well as at 37°C with PRF application for 15 minutes increased as compared to that in cells incubated at 37°C without PRF application. Continuous radio frequency at 70°C but not PRF provoked apoptotic cell death at 1-2 hour, and necrotic cell death at 24 hours after the RF application.

Conclusion: A simple experimental system using human monocytic cells in culture demonstrated that a 15 minute elevation of temperature above 37°C enhanced gene expression for POMC in THP-1 cells, while a 15 minute application of PRF to these cells incubated at 37°C or lower, also enhanced gene expression, indicating that temperature-independent mechanisms as well as thermal effects may be involved in such gene expression.

Keywords: THP-1; apoptosis; apoptotic vesicle; human monocytic cells; necrosis; proopiomela-nocortin; pulsed radio frequency electric field; β-endorphin.

Figure 1

(a1 and a2) Representative FSC-A/SSC-A plots of the suspension of THP-1 cells at 1 hour or 2 hours after the control manipulation, where the active tip of the electric probe from a radio frequency (RF) generator was placed in the sedimented THP-1 cells in a microtube without generating RF currents (No RF). (b1 and b2) Same as a1 and a2 except that the continuous RF current at 70°C for 3 minutes was applied to the sedimented THP-1 cells. (c1 and c2) Same as a1 and a2 except that the pulsed RF current for 15 minutes was applied to the sedimented THP-1 cells at below 43°C. (d–f) The fluorescence of fluorescein isothiocyanate isomer I (FITC) bound to particles detected by flow cytometry was plotted against that of propidium iodide (PI) bound to each particle. FSC-A or SSC-A is the area of electric pulse for the forward scatter (FSC) or the side scatter (SSC), respectively. FITC-A or PI-A is the area of electric pulse for the fluorescence intensity of FITC or PI, respectively. Notes: Particles gated in (−/−) are FITC- and PI-negative and dotted in black in every plot shown in Figure 1. Particles gated in (−/+) (PI-negative/FITC-positive) or (+/+) (PI-positive/FITC-positive) are those of early apoptotic cells (or vesicles) dotted in green, or late apoptotic cells dotted in blue, respectively. Particles gated in (+/−) (PI-positive/FITC-negative) are those of necrotic cells dotted in red. Plots shown as d, e, or f correspond to those shown as a, b, or c, respectively. The CountBright absolute counting beads plotted in a1 and d1 are indicated by arrows. These data were obtained in a series of experiments performed simultaneously and represented at least six separate experiments.

Figure 2

No radio frequency (RF): Representative FSC-A/SSC-A plots of the suspension of THP-1 cells at 24 hours after the control manipulation, where the active tip of the electric probe from a RF generator was placed in the sedimented THP-1 cells in a microtube without generating RF currents. Continuous radio frequency (CRF): Same as “No RF” except that CRF current for 3 minutes was applied to the sedimented THP-1 cells at 70°C. Pulsed radio frequency (PRF): Same as CRF except that the PRF current for 3 minutes, 9 minutes, or 15 minutes was applied to the sedimented THP-1 cells at just below 43°C. CountBright (CB): The CB calibration beads were suspended in the culture medium without adding the suspension of THP-1 cells to confirm the background noise of flow cytometric analysis. The stock suspension of these calibration beads was added to every sample in the same proportion as shown in CB. Particles of propidium iodide (PI)/fluorescein isothiocyanate isomer I (FITC) (−/−) were dotted in black. PI/FITC (−/+) or (+/+) particles are those of early apoptotic cells/vesicles dotted in green, or late apoptotic cells dotted in blue, respectively. PI/FITC (+/−) necrotic cells are dotted in red. These data were obtained in a series of experiments performed simultaneously and represent at least six separate experiments.