Cholera toxin enhances taurine uptake in cultures of human retinal pigment epithelial cells.
Taurine uptake into cultures of human retinal pigment epithelial (HRPE) cells was monitored for 7 days after seeding. A culture medium containing 16% fetal bovine serum (FBS) was used for 2 days and switched to one with 8% FBS. Uptake of taurine (25 nM) was approximately 1.5 pmol/mg protein/15 min for 3 days, then decreased by 45% and was maintained at a decreased level till the 7th day. When the 16% FBS medium was used for the entire culture period, a similar profile of taurine uptake was observed but decrease of the uptake started on the 3rd day. Treatment of cells with 100 ng/ml cholera toxin (CT) for 24 h between the 6th and 7th days returned taurine uptake to its high level observed at the beginning of the cell culture. A similar CT treatment of cells between the 2nd and 3rd days enhanced taurine uptake significantly but this enhancement was much smaller. CT increased taurine uptake in treatment-time and dose dependent manners. Forskolin (FSK) (10 mM) and 8-Bromocyclic adenosine 3',5'-monophosphate (1 mM) also increased taurine uptake. KT5720 at 1 microM, a selective inhibitor of cAMP-dependent protein kinase (PKA), partially blocked CT-induced enhancement of taurine uptake. The level of cAMP was higher on the 3rd day than the 7th day but its response to 3-isobutyl-1-methyl-xanthine, FSK and CT was similar on both days. A kinetic analysis revealed that CT treatment decreases the apparent Michaelis-Menten constant of the taurine transporter while the drastic reduction of taurine uptake during the cell culture period is due to a decrease in the maximal velocity. The results show that cAMP elevated by CT treatment enhances taurine uptake via an increase in the affinity of the transporter. The decrease of taurine uptake during the culture period seems to be related to a decrease in the amount of the transporter.
Miyamoto
Y
Y
School of Optometry, University of California at Berkeley, 94720, USA.
Marczin
N
N
Catravas
J D
JD
Del Monte
M A
MA
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D002227:Carbazoles; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology; D013654:Taurine / Q000378:metabolism*; D000099247:Taurine Transporters
D015056:1-Methyl-3-isobutylxanthine; D015124:8-Bromo Cyclic Adenosine Monophosphate; D002227:Carbazoles; D002352:Carrier Proteins; D002772:Cholera Toxin; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D007211:Indoles; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010726:Phosphodiesterase Inhibitors; D011758:Pyrroles; D013654:Taurine; D000099247:Taurine Transporters; C057416:KT 5720