The cholecystokinin system in the rat retina: receptor expression and in vivo activation of tyrosine phosphorylation pathways.
High levels of endogenous cholecystokinin (CCK) are present in the rat retina and restricted to amacrine cells. Two types of CCK receptors exist but their expression and intracellular transduction pathways coupled in the rat retina are unknown. The aims of this study were to investigate CCK receptors expression in rat retina and to study downstream tyrosine phosphorylation pathways. For this purpose, total mRNA isolated from rat retina was subjected to RT-PCR analysis. Isolated rat retinas were incubated in presence of CCK. Soluble proteins in retinal homogenates were immunoprecipitated with anti-phoshpotyrosine or anti-p130(Cas) specific monoclonal antibodies and subjected to SDS-PAGE, followed by Western blotting analysis. Both types of CCK receptor mRNAs, A and B, are present in the rat retina. Incubation of retina with CCK induced a rapid increase in several phosphotyrosine-containing bands with molecular masses greater than 30 kDa. Western Blotting and immunoprecipitation with a specific monoclonal antibody identified one of the phosphotyrosine bands as the adapter protein p130(Cas). Tyrosine phosphorylation of p130(Cas) induced by CCK in rat retina was time and concentration dependent: CCK induced tyrosine phosphorylation of p130(Cas) occurred rapidly with the maximum effect observed at 2.5 min incubation with 1 microM CCK. Our data clearly identified CCK-A and CCK-B receptor mRNAs in the rat retina and demonstrated that they are functional, stimulating tyrosine phosphorylation pathways. Our results provide novel biochemical information to further understand the physiological role of CCK A and B receptors in rat retina.
Bragado
Maria J
MJ
Department of Physiology, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spain. jbragado@unex.es
Perez-Marquez
Julio
J
Garcia-Marin
Luis J
LJ
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002766:Cholecystokinin / Q000378:metabolism*; D050719:Crk-Associated Substrate Protein; D004586:Electrophoresis; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011506:Proteins; D051381:Rats; D017208:Rats, Wistar; D043886:Receptor, Cholecystokinin A / Q000378:metabolism; D043887:Receptor, Cholecystokinin B / Q000378:metabolism; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D050717:Retinoblastoma-Like Protein p130; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; C493843:Bcar1 protein, rat; D050719:Crk-Associated Substrate Protein; D010750:Phosphoproteins; D011506:Proteins; D043886:Receptor, Cholecystokinin A; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine; D002766:Cholecystokinin