313 Pharmacological Properties of Glycine Transport in the Frog Retina* Jorge A. Pérez-León, 1,2 Estuardo López-Vera, 1,2 and Rocío Salceda 1,3 (Accepted June 12, 2003) The high-affinity glycine transport in neurons and glial cells is the primary means for inactivating synaptic glycine. Two different glycine transporter genes, Glyt-1 and Glyt-2, have been cloned. Glyt-1 has been reported to occur in the retina, but there is no evidence for expression of the Glyt-2 trans- porter. We have pharmacologically characterized glycine transport in the frog retina. 3 H-Glycine uptake in the retina was insensitive to modulation by phorbol esters or changes in cAMP levels, and was par- tially inhibited by sarcosine. Differential sensitivity of glycine transport to sarcosine was exhibited by synaptosomal fractions from the inner and outer plexiform layers of the frog retina. The Na + Hill coef- ficient of glycine uptake was 2.0, as has been reported for Glyt-2. In addition, amoxapine, a specific inhibitor of the Glyt-2a isoform, reduced by 60% glycine uptake by P 2 synaptosomal fraction. Our results indicate the presence of different glycine transporter isoforms in the frog retina, acting mainly through the classical inhibitory glycine system. KEY WORDS: Glycine uptake; retina, glycine transporter1; glycine transporter 2; sarcosine, amoxapine. INTRODUCTION Glycine is considered an inhibitory neurotransmitter in the retina. Biochemical, autoradiographic, and electro- physiological studies have implicated glycine as a retinal neurotransmitter (1–6). An essential property of synaptic transmission is the rapid termination of activity following neurotransmitter release (7). For many neurotransmitters, including glycine, rapid termination of synaptic activity is achieved by a Na + -dependent, high-affinity uptake of the transmitter into the presynaptic terminal and surrounding glial cells (7). Recently, cDNAs encoding glycine transporters have been isolated by different laboratories (Glyt-1 a,b,c and Glyt-2) (8–12) The predicted proteins are members of a super family of Na + -dependent neurotransmitter trans- porters, characterized by 12 putative membrane-spanning segments and cytoplasmic N- and C-terminal extensions containing multiple presumptive phosphorylation sites (10–12). Protein phosphorylation is a central mechanism involved in short-term regulation of cellular processes (13–16). Accordingly, several groups have reported that neu- rotransmitter transporter-proteins are regulated by second messenger-effector systems such as protein kinase C (PKC) (17–19). GABA and glutamate transporters are regulated by phorbol esters acting through PKC (20–22). Similarly, glycine uptake by the Glyt-1 b has been reported to be modulated by activation of PKC (23,24). All Glyt-1 transporter isoforms are pharmacologically distinguishable from Glyt-2 by the sensi- tivity of Glyt-1 isoforms to inhibition by sarcosine (11,25). The presence of Glyt-1 in retina has been demonstrated by immunohistochemical studies (26–29). However a sys- tematic study has not been performed for its characterization. 0364-3190/04/0100–0313/0 © 2004 Plenum Publishing Corporation Neurochemical Research, Vol. 29, No. 1, January 2004 (© 2004), pp. 313–318 * Special issue dedicated to Dr. Herminia Pasantes-Morales. 1 Departamento de Neurociencias, Instituto de Fisiología Celular, UNAM. México, D.F. 2 Current address: Instituto de Neurobiología, UNAM Juriquilla, Qro. México. 3 Address reprint requests to: Instituto de Fisiología Celular, Departa- mento Neurociencias, Universidad Nacional Autónoma de México, Apdo. Postal 70–253, C.P. 04510, México, D.F. México, Tel: 5255-56- 22-56-69; Fax: 5255-56-22-56-07; E-mail: rsalceda@ifisiol.unam.mx