LocalizationofocularP2Y2receptorgeneexpressionbyinsituhybridizationMatthewS.Cowlena,VivianZ.Zhanga,LisaWarnockb,CarolynF.Moyerb,WardM.Petersona,BenjaminR.Yerxaa,*aInspirePharmaceuticals,Inc.,4222EmperorBlvdSuite470,Durham,NC27703,USAbPathologyAssociates,Frederick,MD,USAReceived14August2002;acceptedinrevisedform4March2003AbstractTheobjectiveofthisstudywastodeterminethecellularlocalizationofP2Y2receptorgeneexpressioninrabbitandprimateoculartissuesusingthetechniqueofnon-isotopicinsituhybridization.FreshfrozenwholeeyefromaNewZealandwhiterabbitandwholeeyeandeyelidfromarhesusmacaquewerecutinto5mmthicksectionsandmountedontoglassslides.Insituhybridizationwasperformedonocularcryosectionsusingdigoxigenin-labeledP2Y2receptorriboprobes.Alkalinephosphatase-conjugatedanti-digoxigeninantibodywasusedtolocalizeriboprobehybridization,whichwassubsequentlyvisualizedbystainingwithaprecipitatingalkalinephosphatasesubstrate.CytoplasmicstainingindicativeofantisenseriboprobehybridizationtoP2Y2receptormRNAwasobservedinthepalpebralandbulbarconjunctivalepithelium,includinggobletcells,thecornealepithelium,andinmeibomianglandsebaceousandductalcells.Stainingwasalsoobservedinbothlayersoftheciliarybodyepithelium,subcapsularepitheliumofthelens,andcornealendothelium.Intheposterioreye,stainingwasobservedinvariouslayersoftheretina,includingganglioncell,innernuclear,innersegmentandretinalpigmentepitheliumlayers,intheopticnervehead,andinavarietyofstructureswithinthechoroid.Nospecificstainingofsenseriboprobewasseenonanyoftheocularstructures.TheseresultsdemonstratethattheP2Y2receptorgeneisexpressedinavarietyofocularcellstypesandsuggestthatP2Y2receptorsareassociatedwithdiversephysiologicalfunctionsthroughouttheeye.q2003ElsevierScienceLtd.Allrightsreserved.Keywords:P2Y2receptors;nucleotides;eye;geneexpression;insituhybridization1.IntroductionTheP2Y2receptorisamemberoftheP2YreceptorfamilyofGprotein-coupledreceptorsandrespondswithsimilaraffinitiestotheextracellularnucleotideagonistsadenosine50-triphosphate(...
