Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro.
Since in juvenile rats the liver is the predominant site of erythropoietin (EPO) gene expression, we have used primary cultures of juvenile rat hepatocytes to establish and in vitro system for investigation of oxygen-dependent EPO formation. When isolated hepatocytes were incubated at reduced oxygen...
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| Format: | Journal article |
| Language: | English |
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1993
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| _version_ | 1797081399778344960 |
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| author | Eckardt, K Pugh, C Ratcliffe, P Kurtz, A |
| author_facet | Eckardt, K Pugh, C Ratcliffe, P Kurtz, A |
| author_sort | Eckardt, K |
| collection | OXFORD |
| description | Since in juvenile rats the liver is the predominant site of erythropoietin (EPO) gene expression, we have used primary cultures of juvenile rat hepatocytes to establish and in vitro system for investigation of oxygen-dependent EPO formation. When isolated hepatocytes were incubated at reduced oxygen tensions for 18-48 h, we found increased secretion of EPO protein and elevated levels of EPO mRNA, as determined by RNas protection. This increase was maximal at 3% O2, where EPO mRNA levels after 18 h were approximately 15-fold higher than at 20% O2. The increase in EPO mRNA at low oxygen tensions was specific insofar as [3H]uridine incorporation, as a measure of total RNA synthesis, was reduced by approximately 50% at 3% O2, and it appeared to involve gene transcription since it was abolished in the presence of actinomycin D (35 microM). Significant increases in EPO mRNA were also observed in cells kept at 20% oxygen in the presence of cobalt chloride (50 microM) and nickel chloride (400 microM), but EPO mRNA levels achieved under these conditions were less than 7% of those in cells incubated at 3% oxygen. No increase in EPO mRNA levels was observed in cultures incubated at 20% O2 in the presence of cyclic dibutyryl-AMP (10 microM-3 mM), cyclic 8-bromoGMP (10 microM-1 mM), cyclohexyladenosine (1 microM), 5'-N-ethylcarboxamidoadenosine (1 microM) and phorbol 12-myristate 13-acetate (3 nM). In the presence of 10% carbon monoxide, used to block haem proteins in their oxy conformation, EPO mRNA levels in hepatocytes incubated at low oxygen tensions were reduced to 63%.(ABSTRACT TRUNCATED AT 250 WORDS) |
| first_indexed | 2024-03-07T01:13:57Z |
| format | Journal article |
| id | oxford-uuid:8e011766-e453-4466-ae51-8ad864ef1478 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T01:13:57Z |
| publishDate | 1993 |
| record_format | dspace |
| spelling | oxford-uuid:8e011766-e453-4466-ae51-8ad864ef14782022-03-26T22:54:47ZOxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:8e011766-e453-4466-ae51-8ad864ef1478EnglishSymplectic Elements at Oxford1993Eckardt, KPugh, CRatcliffe, PKurtz, ASince in juvenile rats the liver is the predominant site of erythropoietin (EPO) gene expression, we have used primary cultures of juvenile rat hepatocytes to establish and in vitro system for investigation of oxygen-dependent EPO formation. When isolated hepatocytes were incubated at reduced oxygen tensions for 18-48 h, we found increased secretion of EPO protein and elevated levels of EPO mRNA, as determined by RNas protection. This increase was maximal at 3% O2, where EPO mRNA levels after 18 h were approximately 15-fold higher than at 20% O2. The increase in EPO mRNA at low oxygen tensions was specific insofar as [3H]uridine incorporation, as a measure of total RNA synthesis, was reduced by approximately 50% at 3% O2, and it appeared to involve gene transcription since it was abolished in the presence of actinomycin D (35 microM). Significant increases in EPO mRNA were also observed in cells kept at 20% oxygen in the presence of cobalt chloride (50 microM) and nickel chloride (400 microM), but EPO mRNA levels achieved under these conditions were less than 7% of those in cells incubated at 3% oxygen. No increase in EPO mRNA levels was observed in cultures incubated at 20% O2 in the presence of cyclic dibutyryl-AMP (10 microM-3 mM), cyclic 8-bromoGMP (10 microM-1 mM), cyclohexyladenosine (1 microM), 5'-N-ethylcarboxamidoadenosine (1 microM) and phorbol 12-myristate 13-acetate (3 nM). In the presence of 10% carbon monoxide, used to block haem proteins in their oxy conformation, EPO mRNA levels in hepatocytes incubated at low oxygen tensions were reduced to 63%.(ABSTRACT TRUNCATED AT 250 WORDS) |
| spellingShingle | Eckardt, K Pugh, C Ratcliffe, P Kurtz, A Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro. |
| title | Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro. |
| title_full | Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro. |
| title_fullStr | Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro. |
| title_full_unstemmed | Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro. |
| title_short | Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro. |
| title_sort | oxygen dependent expression of the erythropoietin gene in rat hepatocytes in vitro |
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