Molecular and functional mapping of regional differences in P2Y receptor expression in the rat lens.
Extracellular ATP has been shown to mobilize intracellular Ca(2+) in cultured ovine lens epithelial cells and in human lens epithelium, suggesting a role for purines in the modulation of lens transparency. In this study, we characterized the expression profiles of P2Y receptor isoforms throughout the rat lens at both the molecular and the functional levels. RT-PCR indicated that P2Y(1), P2Y(2), P2Y(4) and P2Y(6) are expressed in the lens, while P2Y(12), P2Y(13) and P2Y(14) are not. Immunohistochemistry, using isoform specific antibodies, indicated that the epithelium does not express P2Y(1) and P2Y(2), but that the underlying fiber cells, which differentiate from the epithelial cells, exhibit strong membranous labeling. Although co-expressed in fiber cells, differences in P2Y(1) and P2Y(2) expression were apparent. P2Y(1) expression extended deeper into the lens than P2Y(2), and its expression co-localized with Cx50 gap junction plaques, while P2Y(2) did not. Labeling for P2Y(4) and P2Y(6) receptors were observed in both epithelial cells and fiber cells, but the labeling was predominantly cytoplasmic in nature. While purine agonist (ATP, ADP, UTP and UDP) application to the lens induced mobilization of intracellular Ca(2+) in cortical fiber cells, little to no effect was observed in the anterior and equatorial epithelium. Thus the inability of UTP and UDP to mobilize intracellular Ca(2+) in the epithelium and the predominately cytoplasmic location of P2Y(4) and P2Y(6) suggests that these receptors may represent an inactive pool of receptors that may be activated under non-physiological conditions. In contrast, our results indicated that P2Y(1) and P2Y(2) are functionally active in fiber cells and their differential subcellular expression patterns suggest they may regulate distinct processes in the lens under steady state conditions.
Hu
Rebecca G
RG
Department of Physiology, School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland 1023, New Zealand.
Suzuki-Kerr
Haruna
H
Webb
Kevin F
KF
Rhodes
Jeremy D
JD
Collison
David J
DJ
Duncan
George
G
Donaldson
Paul J
PJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D003459:Crystallins / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods; D007908:Lens, Crystalline / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D003459:Crystallins; D020033:Protein Isoforms; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D002118:Calcium