Expression of mRNA coding for frog urinary bladder urea carrier in Xenopus laevis oocytes

MARTIAL S, IBARRA C, REBELO L, BERTHONAUD V AND RIPOCHE P

Molecular mechanisms of transport

Elsevier Science Publishers B.V.

Año: 1992; p. 299 - 307

Urea movement across the plasma membrane of mammalian kidney collecting ducts plays an important physiological role in maintaining the osmotic pressure in this tissue. Urea transport has also been described in the amphibian urinary bladder, which may therefore be used as a model of the kidney collecting duct. The protein (s) involved in this transport have not yet been isolated and characterized because of the lack of either specific labeled inhibitors of the transport, antibodies raised against the protein, or probes of the gene coding for the carrier. We described here a new approach for studying this protein, using the strategy employed by Hediger et al [1], that is the expression of amphibian urinary bladder mRNA in Xenopus oocytes.50 nl of mRNA (1µg/µl) were injected into experimetnal oocytes, and water into control oocytes. The uptake of urea was measured by a radiotracer technique up to 7 days after injection. Between 2 days and at most 4 days following injection, the Jurea of oocytes injected with mRNA was about 4 times greated than in oocytes injected with water. The urea permeability of the oocytes injected with mRNA was reduce in the presence of the urea analogue, nitrophenyl-thiourea (NPTU) at a concentration of 10-4 M. Jurea was comparable with the Jurea of not-injected oocytes when they were incubated in the presence of 8Br-cAMP (10-4 M) or when injected with mRNA isolated from rat brain, suggesting that the increase of Jurea corresponds to a direct expression of the urea carrier via the injected mRNA.Size fractionation of the mRNA by electrophoresis under denaturing conditions led to the identification of a fraction able to express the urea carrier. In our conditions, the efficient fraction had a size between 6 ant 10 kbases. The injection of this fraction into oocytes increased the Jurea four-fold and this increase was inhibited by NPTU (10-4M). This mRNA fraction identification is a useful starting point for isolating and characterizing the cDNA coding for the urea transporter.