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Ectonucleotidases of the Rabbit Ciliary Body Nonpigmented Epithelium

From the Department of Physiological Science, University of California, Los Angeles, California.

PURPOSE. To investigate the expression of both the message and function of ENPP1 (a member of the ectonucleotide pyrophosphatase/phosphodiesterase family, also known as PC-1), NTPD1 (a member of the ectonucleoside 5'-triphosphate diphosphohydrolase family, CD39), and ecto-5'-nucleotidase (CD73) in rabbit ciliary body nonpigmented epithelial (NPE) cells.

METHODS. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to reveal the presence of mRNAs of ectonucleotidases in NPE cells. Real-time fluorescence ratio imaging of the intact fura-2-loaded NPE cells was used to record changes in the intracellular calcium concentration.

RESULTS. RT-PCR analysis revealed the expression of mRNAs for ENPP1, NTPD1, and ecto-5'-nucleotidase, but not NTPD2 (ecto-ATPase, or CD39L1), in the rabbit NPE cells. The ENPP1 inhibitor pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), and to a lesser degree the nonspecific ectonucleotidase antagonist 6-N,N-diethyl-ß--dibromomethylene-D-adenosine 5-triphosphate (ARL 67156), reduced the [Ca²⁺]_i increase elicited by the combination of acetylcholine (ACh) and cAMP. However, both inhibitors significantly enhanced the [Ca²⁺]_i increase generated by uridine triphosphate (UTP). The ecto-5'-nucleotidase inhibitor ß-meADP significantly diminished the [Ca²⁺]_i increase evoked by ACh+cAMP, but not that generated by UTP. The A₁-specific adenosinergic receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) significantly blocked the response to ACh+cAMP.

CONCLUSIONS. These observations suggest that rabbit NPE cells possess at least three distinct ectonucleotidases capable of catalyzing the stepwise hydrolysis of adenine and pyrimidine nucleotides, as well as cAMP, thus shaping the purinergic-receptor-coupled signaling in these cells.

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