Abstract
Purpose: :
Retinoic acid (RA) is a common signaling molecule that mediates various cell functions, and serves as a neuromodulator in retina. Our previous studies indicate that the GABAc receptor interacts with RA signal pathway through an interaction with the cellular retinoic acid–binding protein (CRABP). Here, we further investigated the cross–talk between GABA and RA signal pathway by examining the modulatory effects of RA on GABAc receptor expression.
Methods: :
A neuroblastoma cell line (SHp5–ρ1) stably expressing human ρ1 subunit was used in this study. The GABA elicited currents were measured using patch–clamp recording techniques. The mRNA levels of GABA ρ1 subunit in neuroblastoma cells were measured with real–time RT–PCR methods. For each reaction, 100 ng of total RNA were used, and a housekeeping gene, GAPDH, served as a control. The expression of the GABAc receptor protein was determined by immunofluorescence. Cells were fixed with 3% formalin for 30 minutes, and incubated with human ρ1 antibody (1:100).
Results: :
Immunostaining revealed significant enhancement of the GABAc receptor ρ1 subunit expression in cells treated with RA (100 nM, 48 hrs) compared with untreated controls. The level of ρ1 RNA in the cells was determined by comparing the threshold cycle with a calibrated standard curve. After adjusting the RNA level for GAPDH expression, RA–treatment elicited a 2.5 + 0.6 (n=3) fold enhancement of ρ1 mRNA compared with control. RA treatment also increases GABAc receptor activity. The averaged whole–cell membrane currents elicited by 10 µM GABA were 444 + 85 pA (n=7) from untreated control cells, compared to 688 + 110 pA (n=7) for RA treated cells. On the other hand, RA–treatment did not alter the GABA sensitivity of the expressed receptor. The EC50 values of GABA–elicited responses were 2.56 µM, and 2.76 µM for RA treated and untreated cells, respectively.
Conclusions: :
RA treatment enhances GABA ρ1 subunit gene transcription and protein expression in the neuroblastoma cells without altering the properties of the receptor. The pathway for this RA modulation of the GABAc receptor is being investigated.
Keywords: gene/expression • inhibitory receptors • signal transduction