Dual roles of diadenosine polyphosphates in corneal epithelial cell migration.
OBJECTIVE
To investigate the influence of diadenosine polyphosphates on the rate of corneal epithelial cell migration.


METHODS
Primary corneal epithelial cell cultures were obtained from New Zealand White rabbits. Immunocytochemical experiments were performed by fixing the cells with 4% paraformaldehyde (PFA) and incubated with cytokeratin 3 primary antibody, which was subsequently incubated with a secondary IgG mouse labeled with FITC, and the cells were observed under confocal microscopy. Migration studies were performed by taking confluent monolayers that were wounded with a pipette tip and challenged with different di- and mononucleotides with or without P2 antagonist (n = 8 each treatment). For concentration-response analysis, compounds were tested in doses ranging from 10(-8) to 10(-3) M (n = 8). The stability of the dinucleotides was assayed by HPLC, with an isocratic method (n = 4).


RESULTS
Cells under study were verified as corneal epithelial cells via the immunocytochemical analysis. Cell migration experiments showed that Ap4A, UTP, and ATP accelerated the rate of healing (5, 2.75, and 3 hours, respectively; P < 0.05; P < 0.001), whereas Ap3A, Ap5A, and UDP delayed it (6.5, 10, and 2 hours, respectively; P < 0.05). ADP did not modify the rate of migration. Antagonists demonstrated that Ap4A and Ap3A did activate different P2Y receptors mediating corneal wound-healing acceleration and delay. Concerning the possible degradation of the dinucleotides, it was almost impossible to detect any products resulting from their cleavage.


CONCLUSIONS
Based on the pharmacological profile of all the compounds tested, the two main P2Y receptors that exist in these corneal cells are a P2Y(2) receptor accelerating the rate of healing and a P2Y6 receptor that delays this process.
Mediero
Aránzazu
A
Departamento de Bioquímica y Biología Molecular IV, Universidad Complutense de Madrid, Madrid, Spain.
Peral
Assumpta
A
Pintor
Jesús
J
D000818:Animals; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D015226:Dinucleoside Phosphates / Q000494:pharmacology / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000166:cytology* / Q000187:drug effects; D019084:Fluorescent Antibody Technique, Indirect; D007633:Keratins / Q000378:metabolism; D018613:Microscopy, Confocal; D009711:Nucleotides / Q000502:physiology; D058919:Purinergic P2 Receptor Antagonists; D011817:Rabbits; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D014945:Wound Healing / Q000187:drug effects
D015226:Dinucleoside Phosphates; D009711:Nucleotides; C546386:P2ry2 protein, mouse; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C099845:purinoceptor P2Y6; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; C020733:diadenosine tetraphosphate; C011440:diadenosine triphosphate; D007633:Keratins