EXPRESSION OF A RABBIT RENAL ASCORBIC-ACID TRANSPORTER IN XENOPUS-LAEVIS OOCYTES

We examined the expression of renal ascorbic acid transporter(s) in Xe nopus 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. Injectio n of poly(A)+ RNA into oocytes produced over a fivefold increase in th e uptake of [C-14]ascorbic acid (570 mu M) compared with water-injecte d cells. Size fractionation of the kidney cortex mRNA by sucrose gradi ent revealed that the mRNA species that induced ascorbic acid transpor ter 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 asco rbic acid transporter(s) was noticeable as early as 2 days after injec tion and was maximal after 7 days; it was also dependent on the amount of mRNA injected into oocytes. The induced uptake of [C-14]ascorbic a cid after injection of mRNA into oocytes was 1) Na+ dependent, as indi cated by the almost complete lack of transport on removal of Na+ from the incubation medium; 2) significantly inhibited by unlabeled ascorbi c acid and its structural analogue isoascorbic acid but not by D-gluco se; and 3) saturable as a function of increasing the substrate concent ration in the incubation medium (100-1,000 mu M), with an apparent K-m of 258 +/- 72.5 mu M and a maximum velocity of 29.6 +/- 2.8 pmol.oocy te(-1).2 h(-1). These data demonstrate that X. laevis oocytes are a su itable system to functionally express the mammalian renal ascorbic aci d transporter. Furthermore, because of the similar properties of Na+ d ependence, substrate specificity, and kinetics, the expressed ascorbic acid transporter appears to be that of the brush-border membrane of t he renal reabsorptive cells, where it represents a major reabsorptive mechanism.