Extracellular glutathione peroxidase mRNA and protein in human cell lines.

Extracellular glutathione peroxidase (E-GPx) and cellular glutathione peroxidase (C-GPx) are selenoenzymes encoded by two distinct genes. Using specific immunoprecipitations of [75Se]selenium metabolically labeled human cell lines in culture, it was found that Caco-2, Hep3B, Hep G2, and Caki-2 synthesize C-GPx and E-GPx and secrete E-GPx. HBL-100, BT-20, and MCF-7 synthesize only C-GPx. The relationship between Se status (as determined by C-GPx activity) and E-GPx and C-GPx mRNA steady-state levels was investigated in Hep G2, Caco-2, and Caki-2. The most Se-deficient Hep G2, Caco-2, and Caki-2 cells had 8.7 +/- 2.6, 11.2 +/- 4.9, and 9.4 +/- 5.0%, respectively, of C-GPx activity of the replete cells. The steady-state levels of mRNA were measured by Northern and slot blot hybridization analysis. By Northern analysis, a single band was present at 1.0 and 1.80 kb for C-GPx and E-GPx mRNA, respectively, in all three cell lines. Scanning densitometry of the blots revealed that the most Se-deficient cells had 30-50% C-GPx mRNA and 60-80% E-GPx mRNA of the replete cells. It is concluded that, in addition to previously examined human cell lines, Hep3B and Caco-2 make and secrete E-GPx while HBL-100 and BT-20 do not. The slightly reduced levels of G-GPx and E-GPx mRNA in Se-deficient human cell lines can only partially account for the decreased C-GPx activity in Se-deficient human cell lines.

Human kidney proximal tubules are the main source of plasma glutathione peroxidase.

The sites of synthesis of extracellular (E) glutathione peroxidase (GPX), a unique selenoglycoprotein present in plasma, are not known. To investigate the possibility that the kidney is the main source for the plasma GPX, we examined GPX activities and selenium concentrations in the plasma of patients with renal failure on dialysis and nephrectomized patients before and after kidney transplantation. Plasma GPX activities in these patients were 42, 22, and 180% of normal EGPX activity, respectively, whereas plasma Se levels were within the normal range. Twenty-four hours after nephrectomy of anesthetized rats, plasma GPX activity was 30.0 +/- 6.4% of the activity at zero time. Northern hybridization analysis of eight human tissues probed with EGPX and cellular glutathione peroxidase (CGPX) cDNA revealed that the ratio of EGPX to CGPX was highest in the kidney. cRNA in situ hybridization studies on kidney slices showed that only proximal tubular epithelial cells and parietal epithelial cells of Bowman's capsule contained EGPX transcripts. Caki-2, a proximal tubular renal carcinoma cell line, makes and actively secretes EGPX. Taken together, these results strongly suggest that kidney proximal tubular cells are the main source for GPX activity in the plasma.

Sulfation of minoxidil by human liver phenol sulfotransferase.

The N,O-sulfate of minoxidil (Mnx) is the active agent in producing the vasodilation and the hair-growth stimulating responses observed with Mnx treatment. In this report, Mnx sulfation activity was assayed in cytosol prepared from several normal human livers, and Mnx sulfation was shown to correlate significantly with the activity of the phenol-sulfating form of phenol sulfotransferase (P-PST) activity in the same livers. No correlation was observed between Mnx sulfation and the dopamine or dehydroepiandrosterone (DHEA) sulfotransferase activities present in human liver. Mnx sulfation also copurified with P-PST activity during the purification of P-PST from human liver. During the purification procedure, Mnx and p-nitrophenol sulfotransferase (P-PST) activities were resolved from the dopamine and DHEA sulfation activities catalyzed by the monoamine-sulfating form of phenol sulfotransferase (M-PST) and DHEA sulfotransferase respectively. Also, purified DHEA sulfotransferase was not capable of sulfating Mnx, and no data were obtained to indicate that Mnx is a substrate for M-PST. p-Nitrophenol, a substrate for P-PST, was demonstrated to be a competitive inhibitor of Mnx sulfation catalyzed by purified P-PST when Mnx was the variable substrate. These results indicate that Mnx is sulfated and, therefore, bioactivated by P-PST in human liver.