1、Localization of ocular P2Y2receptor gene expressionby in situ hybridizationMatthew S.Cowlena,Vivian Z.Zhanga,Lisa Warnockb,Carolyn F.Moyerb,Ward M.Petersona,Benjamin R.Yerxaa,*aInspire Pharmaceuticals,Inc.,4222 Emperor Blvd Suite 470,Durham,NC 27703,USAbPathology Associates,Frederick,MD,USAReceived
2、14 August 2002;accepted in revised form 4 March 2003AbstractThe objective of this study was to determine the cellular localization of P2Y2receptor gene expression in rabbit and primate ocular tissuesusing the technique of non-isotopic in situ hybridization.Fresh frozen whole eye from a New Zealand w
3、hite rabbit and whole eye and eyelidfrom a rhesus macaque were cut into 5 mm thick sections and mounted onto glass slides.In situ hybridization was performed on ocularcryosections using digoxigenin-labeled P2Y2receptor riboprobes.Alkaline phosphatase-conjugated anti-digoxigenin antibody was used tol
4、ocalize riboprobe hybridization,which was subsequently visualized by staining with a precipitating alkaline phosphatase substrate.Cytoplasmic staining indicative of antisense riboprobe hybridization to P2Y2receptor mRNA was observed in the palpebral and bulbarconjunctival epithelium,including goblet
5、 cells,the corneal epithelium,and in meibomian gland sebaceous and ductal cells.Staining was alsoobserved in both layers of the ciliary body epithelium,subcapsular epithelium of the lens,and corneal endothelium.In the posterior eye,staining was observed in various layers of the retina,including gang
6、lion cell,inner nuclear,inner segment and retinal pigment epitheliumlayers,in the optic nerve head,and in a variety of structures within the choroid.No specific staining of sense riboprobe was seen on any of theocular structures.These results demonstrate that the P2Y2receptor gene is expressed in a
7、variety of ocular cells types and suggest that P2Y2receptors are associated with diverse physiological functions throughout the eye.q 2003 Elsevier Science Ltd.All rights reserved.Keywords:P2Y2receptors;nucleotides;eye;gene expression;in situ hybridization1.IntroductionThe P2Y2receptor is a member o
8、f the P2Y receptorfamily of G protein-coupled receptors and responds withsimilar affinities to the extracellular nucleotide agonistsadenosine 50-triphosphate(ATP)and uridine 50-triphosphate(UTP)(Ralevic and Burnstock,1998).Based on pharma-cological and physiological characterizations,P2Y2orP2Y2-like
9、 receptors have been found on many diverse celltypes,including epithelial,neuronal,glial,bone(osteoclastsand osteoblasts),and endothelial cells(Burnstock andWilliams,2000).Cloned and endogenous P2Y2receptorsin a number of systems have been shown to signal throughGq/11and Gi/oproteins and stimulate v
10、arious intracellularsignalling effector molecules and proteins to elicit amultitude of cell type-specific responses(Dubyak andel-Moatassim,1993).For example,Gq-mediated activationinitiates a well-defined cascade of intracellular signallingevents comprising activation of phospholipase C-b,for-mation
11、of inositol trisphosphate,release of Ca2fromintracellular stores,and influx of Ca2from the plasmamembrane into the cytosol.P2Y2receptor-dependentactivation has also been shown to stimulate other effectorsignalling molecules,including protein kinase C,phos-pholipase A2,Ca2-dependent Kchannels,phospho
12、lipaseD,mitogen-activated protein kinases,and stress-activatedprotein kinases(Ralevic and Burnstock,1998).Given thisdiversity of functional expression and stimulation ofintracellular signalling pathways,P2Y2receptor activationis linked to multiple physiological roles in different cell-andtissue-spec
13、ific systems.0014-4835/03/$-see front matter q 2003 Elsevier Science Ltd.All rights reserved.DOI:10.1016/S0014-4835(03)00068-XExperimental Eye Research 77(2003) author.Dr Benjamin R.Yerxa,Inspire Pharmaceuticals,Inc.,4222 Emperor Blvd,Suite 470,Durham,NC 27703,USA.E-mail address:(B.R.Yerxa).This div
14、ersity in P2Y2receptor signalling and function isapparent in the eye(Pintor,1999).Using a variety ofpharmacological and physiological approaches,the pre-sence of receptors that are pharmacologically consistentwith P2Y2receptors has been identified in cornealepithelium and endothelium(Srinivas et al.
15、,1998;Kimuraet al.,1999;Cha et al.,2000;Yang et al.,2000),conjunctival epithelium(Jumblatt and Jumblatt,1998;Hosoya et al.,1999;Shiue et al.,2000;Li et al.,2001b),ciliary epithelia(Wax et al.,1993;Shahidullah and Wilson,1997),Mu ller cells(Liu and Wakakura,1998;Bringmannet al.,2002),retinal pigment
16、epithelium(Sullivan et al.,1997)and ganglion cells(Wheeler-Schilling et al.,2001),supracoroid(Sugamoto et al.,1999),and epithelial cells inthe lens(Collison and Duncan,2001).These publishedresults clearly suggest that P2Y2-like receptors regulatemultiple cellular functions in ocular physiology.Forex
17、ample,natural and synthetic P2Y2receptor agonistsstimulate ion and fluid secretion across intact conjunctivalepithelia and mucin discharge from conjunctival gobletcells,and therefore may be useful to lubricate the ocularsurface in dry eye disease(Murakami et al.,2000a,b).INS365,a synthetic agonist f
18、or this receptor,is showingpromise as a novel therapeutic for the treatment of dry eyedisease(Mundasad et al.,2001),and has recently completedPhase 3 testing in dry eye patients in the United States.Activation of P2Y2receptors by UTP and the syntheticagonist INS37217 in the RPE stimulates net apical
19、-to-basolateral fluid absorption in vitro,and INS37217 has beenshown to stimulate subretinal fluid reabsorption in vivo,presumably by activating the RPE fluid pump(Petersonet al.,1997;Blaug,2000;Maminishkis et al.,2000).Stimulation of subretinal fluid reabsorption by INS37217 iscurrently being explo
20、red in the clinic as a potentialtreatment for some forms of retinal detachment,whichoccurs as a result of pathological accumulation of fluid inthe subretinal space.Despite the number of functional and pharmacologicaldata on P2Y2receptors in the eye,very little of this work hasfocusedongeneorproteine
21、xpressionoftheP2Y2receptorinspecific cell types.In the present study,we mapped thelocalization of P2Y2receptor mRNA using in situ hybridi-zation(ISH)techniques in ocular tissues of the rhesusmacaqueandtheNewZealandwhiterabbit.Ourresultsofferdefinitive localization of P2Y2receptor mRNA in variousocul
22、ar tissues,including some not previously reported toexpress this receptor,and additionally offer a direct com-parison of receptor expression in two mammalian species.2.Materials and methods2.1.TissuesEyes were obtained from a New Zealand white rabbit anda rhesus macaque;eyelids were obtained from th
23、e monkeyonly.Lung(positive control tissue)was obtained from bothspecies.All tissues were provided by Pathology Associates(Frederick,MD).Tissues were removed from healthyanimals immediately following euthanasia and snap frozenin embedding medium.Frozen tissues were stored at 2808Cprior to cryosection
24、ing.Tissues were cut in 5 mm sectionsand mounted on SuperFrost Plus microscope slides(FisherScientific,Pittsburgh,PA)for hematoxylin and eosin(H andE)staining and in situ hybridization.Sections stained withHand E were prepared to evaluate the quality and orientationof the study tissues.Examination o
25、f H and E slides indicatedthat all tissues were suitable for ISH.Slide-mounted tissuesections were kept frozen until all sections were cut,thenused immediately for ISH.2.2.Riboprobe synthesisRiboprobes for this study were synthesized fromnucleotides 272627 of the human P2Y2receptor gene.The DNA sequ
26、ence of this fragment of the human gene wasfound to be 96%homologous with the correspondingfragment of the monkey P2Y2receptor gene.Although thecorresponding fragment from the rabbit was not cloned andsequenced,northern blot analysis of rabbit lung RNAdemonstrated that the human riboprobe hybridizes
27、 withrabbit P2Y2mRNA under stringent conditions.Template DNA for riboprobe synthesis was obtained byPCR amplification using primers designed to incorporateeither an upstream T3 promotor or a downstream T7promotor.The resulting PCR products were used tosynthesize digoxigenin-labeled riboprobes by in
28、vitrotranscription(IVT).Antisense and sense riboprobes weresynthesized using T7 and T3 RNA polymerases,respect-ively,in the presence of digoxigenin-11-UTP(RocheMolecular,Indianapolis,IN)using a MAXIscript IVT kit(Ambion,Austin,TX)according to the manufacturersinstructions.Following IVT,template DNA
29、was degradedwith DNase-1,and unincorporated digoxigenin wasremoved by centrifugal ultrafiltration using Microconcolumns(Millipore,Bedford,MA).Riboprobe integritywas assessed by electrophoresis through a denaturingpolyacrylamide gel.Apparent molecular size was estimatedby comparison with the electrop
30、horetic mobility of a1001000 base pair RNA ladder(Ambion).Probe yieldand labelling was evaluated by blot immunochemistry.Riboprobes were dispensed in 5 ml aliquots and stored at2808C until used for ISH.2.3.In situ hybridizationFrozen sections were fixed in 4%paraformaldehyde inPBS,pH 74,for 15 min a
31、t room temperature.TissueRNases were inactivated by treatment with 01%diethylpyrocarbonate for 30 min.Sections were incubated over-night in hybridization buffer(50%formamide,5X SSC,40 mg ml21sheared salmon sperm DNA)containingM.S.Cowlen et al./Experimental Eye Research 77(2003)77847805 mg ml21of eit
32、her antisense or sense probe.Followinghybridization,slides were subjected to a series of highstringency washes to reduce non-specific hybridization.Hybridization was visualized by immunohistochemistryusing an alkaline phosphatase-conjugated anti-digoxigeninantibody and the precipitating alkaline pho
33、sphatase sub-strate nitroblue tetrazolium chloride/bromochloroindolylphosphate(Roche Molecular)according to the manufac-turers instructions.Tissue sections were counter stainedwith nuclear fast red for 2 min,rinsed in deionized water,and coverslipped.Assay controls included omission ofprobe and omis
34、sion of probe and anti-digoxigenin antibody.2.4.Evaluation of stainingCells were assessed for demonstration of hybridizationwith the antisense P2Y2receptor probe by visualizing darkbrown/black cytoplasmic and/or peri-nuclear stainingindicative of a positive ISH signal.Each cell type wascompared to r
35、eplicate sections that were hybridized with thesense P2Y2receptor probe.Results were considered positiveif staining was observed with the antisense probe,and nostaining or weak background staining was observed with thesense probe.Microscopic evaluation and image acquisitionwere conducted using an Ol
36、ympus BX51 microscope(Olympus America,Inc.,Melville,NY)and a SPOT digitalcamera(Diagnostic Instruments,Inc.,Sterling Heights,MI)with Adobe Photoshop software(Adobe Systems,Inc.,SanJose,CA).Input levels were adjusted identically betweencorresponding antisense and sense probe images to improveimage co
37、ntrast and appearance.No other digital alterationswere made.3.ResultsNon-radioisotopic ISH was used to determine the cellularlocalization of P2Y2receptor gene expression in themammalian eye(albino rabbit and monkey)and eyelid(monkey).Prior to performing experiments with oculartissues,the non-radiois
38、otopic ISH method was validated in apositive control tissue.Previous results with radioisotopicISH have demonstrated P2Y2receptor gene expression inhuman lung epithelial cells(Shaffer et al.,1998).Therefore,monkey and rabbit lung were chosen as the positive controltissues for P2Y2receptor expression
39、(Table 1).ISH staining,indicative of P2Y2receptor gene expression,was observedin bronchial epithelium,including goblet cells,and insubmucosal gland epithelium,but not in peribronchialsmooth muscle or stromal cells.No staining was observed inany cell type with the P2Y2receptor sense probe.Similarto t
40、he results observed in the monkey,rabbit lung exhibitedintense cytoplasmic staining in airway epithelial cells,including goblet cells,but not in stromal elements.Theresults in monkey and rabbit lung are consistent with theresultsreportedpreviouslyforhumanlungusingradioisotopic ISH,and provide strong
41、 support that thenon-radioisotopic ISH technique used in this study providedsensitive and specific detection of P2Y2mRNA in oculartissues.Representative photomicrographs of ISH results frommonkey ocular tissues are presented in Figs.16.Negativecontrol sense probe images are shown to the right ofanti
42、sense images of each tissue for comparison;no stainingwas observed in any cell type in the eye or eyelid of eitherspecies with the P2Y2sense riboprobe.Photomicrographs ofthe ciliary body and retina from the albino rabbit exhibitingnon-pigmented epithelium are presented in Figs.4 and 5 toallow for co
43、mparison with pigmented epithelium in themonkey.Images of other rabbit oculartissues are notshown.Results from all tissues from both species are summarized inTable 2.ISH staining was observed in the palpebral conjunctiva,the epithelium of the lid margin,and the meibomian glandin monkey eyelid(Fig.1)
44、.Staining was observed in gobletcells of the palpebral conjunctiva,and in modifiedsebaceous units of the meibomian gland and meibomiangland ductal epithelium.Neighbouring nerve cells andstroma were negative for P2Y2gene expression with theantisense probe in monkey eyelid.In monkey and rabbiteye,stro
45、ng staining for P2Y2mRNA was observed in thecorneal epithelium and adjacent bulbar conjunctiva,and inbulbar conjunctival goblet cells when examined at highermagnification,butnotinbulbarconjunctival stroma(Fig.2).Staining was also observed in the corneal endothelium,butnot in Bowmans membrane,Desceme
46、ts membrane,orcorneal stroma.In the lens,ISH signal was observed in thesubcapsular epithelium,but not in the lens stroma or lenscapsule(Fig.3).Assessment of ciliary body epithelium,wascomplicated by the presence of pigmented cells in monkeyeye.Nevertheless,staining for P2Y2receptor mRNA wasdiscernab
47、le in the pigmented cell layer,and was clearlyobserved in the non-pigmented cell layer,in monkey ciliaryepithelium with the antisense probe(Fig.4(A).Stainingwas clearly evident in both cell layers of the ciliaryepithelium in the albino rabbit(Fig.4(C).No stainingTable 1Cellular localization of P2Y2r
48、eceptor gene expression in monkey andrabbit lung(positive control tissues)as determined by non-radioisotopic insitu hybridizationTissueResultLungBronchial epithelium1,2Goblet cells1,2Peribronchial smooth muscle1,32Stroma1,22Submucosal glands1,3Vascular endothelium1,31Monkey.2Rabbit.3Not evaluated in
49、 rabbit.M.S.Cowlen et al./Experimental Eye Research 77(2003)778479was observed in the stroma of the ciliary body in eitherspecies.A comparison of staining in the iris epitheliumbetween monkey and rabbit was not possible due to highlevels of pigment in the monkey.Staining was observed inthe iris epit
50、helium of the rabbit(not shown).In the retina,strong staining for P2Y2mRNA wasobserved with the antisense probe in the ganglion cell layerin both the monkey(Fig.5(A)and the albino rabbit(Fig.5(C).Despite the presence of pigment granules,P2Y2receptor gene expression was discernable in the retinalpigm
