Identification of a Novel Metabotropic Glutamate Receptor (mGluR1) Splice Variant mGluR1b and a Vesicular Glutamate Transporter 1 (VGLUT1) That Defines a Glutamatergic Phenotype in the Human Ciliary Epithelium

Abstract

Purpose:: Glutamate has many important physiological functions including its role as neurotransmitter in the retina and in the central nervous system (CNS). However, in recent years it has been found that glutamate signaling is not restricted to neural tissues. It has been found in numerous non-neuronal tissues including the gastrointestinal tract, bone cells, testes, and in the retinal pigment epithelium. The purpose of this work is to characterize a glutamatergic phenotype in the human ocular ciliary epithelium.

Methods:: RT-PCR amplification from polyA+-mRNA selected from dissected human ciliary processes (CP) and cultured ciliary nonpigmented epithelial cells (NPE), Northern blot and 3’-Rapid Amplification of cDNA Ends (RACE). Oligonucleotide primer pairs were selected for the following cDNAs: mGluR1, glutamate transporters (GLAST, GLT-1 and VGLUT1), glutamine synthase and glutaminase. The cellular distribution and protein expression of VGLUT1 were assessed by immunofluorescence microscopy and Western blot respectively using a VGLUT1 antibody (Cat#135 311, Synaptic Systems, Göttingen, Germany).

Results:: By RT-PCR and DNA sequencing we confirmed that the human CP expresses mRNAs for mGluR1, GLAST, GLT-1, VGLUT1, and glutamate metabolizing enzymes. A radiolabeled DNA probe (474-bp) prepared by RT-PCR for mGluR1 hybridized by Northern blotting to a 7.5-kb transcript in the retina, and to a 6 to 6.5-kb size transcript in ciliary processes (CP). 3’-RACE of the smaller mGluR1 transcript in the CP revealed to correspond to the splicing variant mGluR1b. This splice variant contains an 85n insertion at the intracellular C-terminus, between nucleotides 2660 and 2661 of the wild type mGluR1. The insertion is in frame, but introduces a premature stop codon (TGA) within this extra 85n that results in a replacement of the 313 C-t residues by only 20 residues. By Western blot a VGLUT1 antibody recognized a major band of Mr=60kDa on microsome fractions from retina, CP and NPE cells. By indirect immunofluorescence the VGLUT1 antibody labeled NPE cells in cryostat sections of the ciliary epithelium and in cultured NPE cells.

Conclusions:: VGLUT1 is both necessary and sufficient for uptake and storage of glutamate and thus comprises the sole determinant for glutamatergic phenotype. Overall, these findings suggested that the human ciliary epithelium expresses a glutamatergic system.

Keywords: inflow/ciliary body • excitatory amino acid receptors • excitatory neurotransmitters