Expression of a rabbit renal ascorbic acid transporter in Xenopus laevis oocytes.
We examined the expression of renal ascorbic acid transporter(s) in Xenopus laevis oocytes after microinjection of cells with poly(A)+ RNA extracted from rabbit kidney cortex. Concomitant expression of the Na+-glucose cotransporter served as a control in these studies. Injection of poly(A)+ RNA into oocytes produced over a fivefold increase in the uptake of [14C]ascorbic acid (570 microM) compared with water-injected cells. Size fractionation of the kidney cortex mRNA by sucrose gradient revealed that the mRNA species that induced ascorbic acid transporter expression in oocytes was present in a fraction centered around 2.0 kilobases (kb) and had a size range of 1.8-3.1 kb. Injection of the active fraction into oocytes produced a > 40-fold increase in ascorbic acid uptake compared with water-injected controls. Expression of ascorbic acid transporter(s) was noticeable as early as 2 days after injection and was maximal after 7 days; it was also dependent on the amount of mRNA injected into oocytes. The induced uptake of [14C]ascorbic acid after injection of mRNA into oocytes was 1) Na+ dependent, as indicated by the almost complete lack of transport on removal of Na+ from the incubation medium; 2) significantly inhibited by unlabeled ascorbic acid and its structural analogue isoascorbic acid but not by D-glucose; and 3) saturable as a function of increasing the substrate concentration in the incubation medium (100-1,000 microM), with an apparent Km of 258 +/- 72.5 microM and a maximum velocity of 29.6 +/- 2.8 pmol.oocyte-1.2 h-1. These data demonstrate that X. laevis oocytes are a suitable system to functionally express the mammalian renal ascorbic acid transporter.(ABSTRACT TRUNCATED AT 250 WORDS)
Dyer
D L
DL
Veterans Affairs Medical Center, Long Beach 90822.
Kanai
Y
Y
Hediger
M A
MA
Rubin
S A
SA
Said
H M
HM
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000493:pharmacokinetics; D007267:Injections; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D009865:Oocytes / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger; D011817:Rabbits / Q000378:metabolism*; D012964:Sodium / Q000502:physiology; D013997:Time Factors; D014982:Xenopus laevis / Q000378:metabolism*
D002352:Carrier Proteins; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D012964:Sodium; D005947:Glucose; D001205:Ascorbic Acid