Effect of inhibitors of oxidative phosphorylation on erythropoietin mRNA in isolated perfused rat kidneys

Abstract

We have used RNase protection to measure oxygen-dependent changes in erythropoietin (EPO) mRNA in isolated perfused kidneys and to compare the effect of hypoxia with the response to inhibitors of oxidative phosphorylation. In well-oxygenated kidneys perfused for 2 h at 12 ml/min, with hematocrit of 0.09 +/- 0.005 and PO2 of 443 +/- 67 mmHg, EPO mRNA levels were similar to the baseline levels measured in nonperfused contralateral kidneys from the same animals. When perfusions were performed under identical conditions but at a PO2 of 32 +/- 4 mmHg, EPO mRNA increased approximately 16-fold. In contrast, graded concentrations of cyanide (10, 100, and 300 microM and 1 mM), antimycin (0.01, 0.1, 0.5, and 1 microM), and oligomycin (0.01, 0.1, and 1 microM) did not alter EPO mRNA in well-oxygenated perfused kidneys. However, in kidneys perfused at low PO2 with a high concentration of each inhibitor, EPO mRNA levels were increased, demonstrating that the ability to respond to hypoxia was retained. Thus inhibitors of oxidative phosphorylation did not mimic the effects of hypoxia, indicating that oxygen-dependent expression of the EPO gene in the kidney is not effected through hypoxic compromise of oxidative phosphorylation.

Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism.

“…4A). In contrast, but in keeping with the physiological regulation of the Epo gene (14,15), hypoxic stimulation in a23 cells was not mimicked by exposure to cyanide. Thus, cyanide (10-1000 ,uM) did not induce enhancer activity, nor did it abrogate the response to concurrent hypoxic stimulation (Fig.…”

“…Thus, it is activated by cobalt, but not by cyanide, and is abrogated by cycloheximide (5,6,(13)(14)(15). The dose range of cyanide was chosen to include concentrations which cause minimal, partial, and total inhibition of cellular respiration (15,20).…”

Photometric characteristics of haem proteins in erythropoietin-producing hepatoma cells (HepG2)

“…Special attention was given to the differences in sensitivity to hypoxia and cyanide or antimycin of intracellular haem proteins, since it has been reported that Epo production can be stimulated by hypoxia but not by cyanide poisoning (Goldberg et al, 1988) or by other inhibitors of the respiratory chain (Tan and Ratcliffe, 1991). We postulate, therefore, that the putative haem protein responsible for the oxygen-sensing process in HepG2 cells should be sensitive to hypoxia only.…”

Mitochondrial reactive oxygen species trigger hypoxia-induced transcription

“…Mechanisms including NADPH oxidase (9), cytochrome P 450 (10), and an O 2 -binding heme protein (11) have been proposed. Mitochondria have been considered as possible O 2 sensors, but mitochondrial inhibitors failed to activate transcription (12). But recent work has implicated mitochondria in the O 2 sensing underlying functional responses to hypoxia (13).…”