Effects of low-intensity ultrasound on gramicidin D-induced erythrocyte edema.

Abstract

OBJECTIVES: To determine whether low-intensity ultrasound (US) can reduce red

blood cell (RBC) edema and, if so, whether the US activity is associated with

aquaporin 1 (AQP-1), a water channel in the cell membrane. METHODS: Red blood

cell edema was induced by gramicidin D treatment at 40 ng/mL for 20 minutes and

evaluated by a hematocrit assay. Low-intensity continuous wave US at 1 MHz was

applied to RBCs for the last 10 minutes of gramicidin D treatment. To determine

whether US activity was associated with AQP-1, RBCs were treated with 40 muM

mercuric chloride (HgCl(2)), an AQP-1 inhibitor, for 20 minutes at the time of

gramicidin D treatment. Posttreatment morphologic changes in RBCs were observed

by actin staining with phalloidin. RESULTS: Red blood cell edema increased

significantly with gramicidin D at 20 (1.8%), 40 (6.7%), 60 (16.7%), and 80

(11.3%) ng/mL, reaching a peak at 60 ng/mL, compared to the control group (20

ng/mL, P = .019; 40, 60, and 80 ng/mL, P < .001). No significant RBC hemolysis

was observed in any group. Edema induced by gramicidin D at 40 ng/mL was

significantly reduced by US at 30 (3.4%; P = .003), 70 (4.4%; P = .001), and 100

(2.9%; P = .001) mW/cm(2). Subsequent experiments showed that edema reduction by

US ranged from 7% to 10%. Cotreatment with HgCl(2) partially reversed the US

effect and showed a significantly different level of edema compared to gramicidin

D-alone and US-cotreated groups (P = .001). These results were confirmed by

microscopic observation of RBC morphologic changes. CONCLUSIONS: Low-intensity US

could reduce gramicidin D-induced RBC edema, and its effect appeared to at least

partly involve regulation of AQP-1 activity. These results suggest that

low-intensity US can be used as an alternative treatment to control edema and

related disorders.