Endothelial Cells Contain a Glycine-Gated Chloride Channel

Abstract

Glycine inhibited growth of B16 melanoma tumors in vivo most likely because of the inhibition of angiogenesis. Here, the hypothesis that the anticancer effect of glycine in vivo is due to expression of a glycine-gated Cl- channel in endothelial cells was tested. First, the effects of glycine on vascular endothelial growth factor-induced increases in intracellular Ca²⁺ concentration in a bovine endothelial (CPA) cell line were studied. Vascular endothelial growth factor (1 ng/ml) increased intracellular Ca²⁺ concentration, with peak values reaching 141 ± 11 nM. Glycine blunted this increase dose dependently. Furthermore, the inhibitory effects of glycine were prevented by 1 μM strychnine, a glycine receptor antagonist, or when cells were incubated in Cl^--free buffer. Moreover, glycine increased influx of ³⁶Cl into CPA cells ~10-fold; this reaction was also strychnine sensitive. Furthermore, mRNA similar to the β-subunit of the glycine-gated Cl^- channel from spinal cord was identified in endothelial cells by reverse transcription-polymerase chain reaction. In addition, Western analysis using antibody for the glycine receptor demonstrated expression of the β-subunit of the glycine receptor. Importantly, glycine diminished serum-stimulated proliferation and migration of endothelial cells. Collectively, these data indicate that the inhibitory effect of glycine on growth and migration of endothelial cells is due to activation of a glycine-gated Cl^- channel. This hyperpolarizes the cell membrane and blocks influx of Ca²⁺, thereby minimizing growth factor-mediated signaling.