Abstract
Abstract: :
Purpose: Excess glutamate and iron have been implicated in various neural and retinal degenerations; however a relationship between iron and glutamate has not been explored. Glutamate is an excitatory neurotransmitter whose function has been widely studied in neuronal cells. However, its role as a signaling molecule in non–neuronal cells has only recently been investigated. We have evidence that a cytosolic aconitase, regulated by iron, can control the synthesis of glutamate and its release in retinal pigment epithelial cells. In the present study, we demonstrate that primary cultures of canine lens epithelial cells (LEC) express a glutamatergic system that is regulated by iron. Methods: Primary cultures of canine LEC were incubated in glutamine–free, serum–free MEM with or without ferric ammonium citrate (FAC) for 6 and 24 hours after which the cell conditioned media (CCM) was collected. The concentration of glutamate in the CCM was determined by an Amplex Red assay kit (Molecular Probes). The presence of vesicular glutamate transporter 1 (VGLUT1) mRNA was determined by RT–PCR. VGLUT1 protein expression was analyzed by western blotting. Results: At as early as 6 hours, the concentration of glutamate in the control CCM was 30 ± 3.2µM. With the addition of iron in the form of FAC, the glutamate concentration in the CCM was 36 ± 4.3µM (n = 6, p<0.05 compared to control). By 24 hours, glutamate accumulated in the control CCM to a concentration of 66 ± 13.9µM. However, with FAC supplementation, the concentration of glutamate in the CCM increased to 118 ± 21.9µM (n = 6, p<0.05 compared to control). We have also demonstrated that cytosolic aconitase activity is increased with FAC treatment by 52% compared to control. In cells with vesicular release of glutamate, VGLUTs are responsible for loading vesicles with glutamate. The secretion of glutamate in LEC may be mediated through vesicular release. RT–PCR confirmed the presence of VGLUT1 mRNA, but not VGLUT2 or VGLUT3 mRNA in LEC. VGLUT1 protein was detected in the membrane fraction of LEC through western blot analysis. Conclusions: This study demonstrates a link between iron and glutamate secretion. Dysregulation of iron metabolism could result in alterations in glutamate production and deleterious downstream effects resulting in neural and retinal degeneration. The presence of a glutamatergic system in the lens is puzzling and a role for vesicular glutamate secretion in lens and ocular physiology needs to be explored.
Keywords: excitatory neurotransmitters • metabolism