Regulation of expression of taurine transport in two continuous renal epithelial cell lines and inhibition of taurine transporter by a site-directed antibody.
The renal tubular epithelium adapts to changes in the sulfur amino acid composition of the diet, particularly in terms of reabsorption of taurine. The adaptive response is expressed by enhanced or decreased NaCl-dependent taurine transport by rat renal brush border membrane vesicles (BBMV). Taurine transport activity in two cultured renal epithelial cell lines (MDCK and LLC-PK1) is up- or down-regulated by extracellular taurine concentration as the result of reciprocal changes in the Vmax of the transporter. In MDCK cells, abundance of taurine transporter mRNA (pNCT mRNA) was up- or down-regulated after incubation in media containing 0, 50, or 500 microM taurine. Decreased mRNA was observed in both cell lines after 12 h, and it was appreciably reduced after 72 h exposure to 500 microM taurine. Northern blot analysis of mRNA from LLC-PK1 cells using pNCT cDNA as a riboprobe showed that two transcripts, 9.6 kb and 7.2 kb, were expressed; the abundance of mRNA was increased or decreased after incubation in taurine-free or high taurine medium, respectively. Down-regulation was observed primarily in the 7.2 kb transcript after 24 h incubation. Rapid up-regulation occurred in the 9.6 kb transcript within 12 h of transfer from high to low taurine. Nuclear run-off assays showed that the gene for pNCT is induced at the transcriptional level by taurine. Regulation of expression of the taurine transporter was also studied by injection of pNCT cRNA into Xenopus laevis oocytes. Expression of transport activity was significantly reduced (64%) when oocytes were incubated in 50 microM taurine as compared to 0 microM taurine. Transport activity was totally blocked when pNCT cRNA-injected oocytes were exposed to an active phorbol ester, PMA (10(-6) M). Inhibition of uptake was reversed by staurosporine, an inhibitor of protein kinase C activity. An inactive phorbol ester, 4 alpha-phorbol, had no effect on taurine transport. A polyclonal antibody directed a highly conserved intracellular segment between homologous transmembrane domains VI and VII inhibited taurine transport activity in both pNCT cRNA-injected oocytes and BBMV. Incubation of oocytes with 10 micrograms/ml antibody (Ab) reduced taurine uptake to 46% of control, and 20-80 micrograms/ml Ab reduced uptake to 20% of control. In BBMV, active taurine uptake (10 microM) was inhibited approximately 30% by 10 pg Ab/mg protein, whereas none specific IgG had no significant effect. Proline uptake (20 microM) by BBMV was not inhibited by the Ab, nor was GABA uptake (50 microM). Two pNCT proteins, approximately 70 kD and approximately 30 kD, were detected by Western blot, and the abundance of both was regulated by medium taurine.


CONCLUSIONS
(i) regulation of taurine transport activity in LLC-PK1 cells by medium taurine occurs at a level of mRNA transcription; (ii) regulation of pNCT occurs at both transcriptional and translational levels; (iii) pNCT expression is regulated by protein kinase C-dependent phosphorylation; and (iv) the intracellular segment between domains VI and VII may be required for activation of the taurine transporter; this segment may function as a gate in taurine transport.
Han
X
X
Department of Pediatrics, University of Tennessee, Memphis, USA.
Chesney
R W
RW
Budreau
A M
AM
Jones
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000502:physiology; D002460:Cell Line; D017931:DNA Primers; D004285:Dogs; D004848:Epithelium / Q000378:metabolism; D005786:Gene Expression Regulation; D007668:Kidney / Q000378:metabolism*; D007672:Kidney Cortex / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000502:physiology; D026901:Membrane Transport Proteins; D008871:Microvilli / Q000378:metabolism; D009865:Oocytes / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013654:Taurine / Q000378:metabolism* / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014982:Xenopus laevis; D000099247:Taurine Transporters
D000906:Antibodies; D002352:Carrier Proteins; D017931:DNA Primers; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D013654:Taurine; D000099247:Taurine Transporters