Ectonucleotidases of the rabbit ciliary body nonpigmented epithelium.
OBJECTIVE
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-beta-gamma-dibromomethylene-D-adenosine 5-triphosphate (ARL 67156), reduced the [Ca(2+)](i) increase elicited by the combination of acetylcholine (ACh) and cAMP. However, both inhibitors significantly enhanced the [Ca(2+)](i) increase generated by uridine triphosphate (UTP). The ecto-5'-nucleotidase inhibitor alphabeta-meADP significantly diminished the [Ca(2+)](i) increase evoked by ACh+cAMP, but not that generated by UTP. The A(1)-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.
Farahbakhsh
Nasser A
NA
Department of Physiological Science, University of California, Los Angeles, California 90095, USA. farahbal@ucla.edu
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000225:Adenine / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D001081:Apyrase; D002118:Calcium / Q000378:metabolism; D002924:Ciliary Body / Q000201:enzymology*; D000242:Cyclic AMP / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium / Q000201:enzymology; D016257:Fura-2 / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006868:Hydrolysis; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000201:enzymology; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015703:Antigens, CD; D004791:Enzyme Inhibitors; D011742:Pyrimidine Nucleotides; D012333:RNA, Messenger; D000242:Cyclic AMP; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases; D001081:Apyrase; C067869:CD39 antigen; D000225:Adenine; D002118:Calcium; D016257:Fura-2