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Oxygen pressure-dependent control of carbonic anhydrase synthesis in chick embryonic erythrocytes
Insights
Chick embryonic erythrocytes synthesize carbonic anhydrase (CA) in response to a heat-labile plasma factor. This factor stimulates CA synthesis via transcription, suggesting a role in oxygen homeostasis during development.
Area of Science:
- Biochemistry
- Developmental Biology
- Physiology
Background:
- Carbonic anhydrase (CA) expression in chick erythrocytes is low until the final week of embryonic development.
- Hypoxia is a suspected physiological stimulus for the rapid increase in CA synthesis before hatching.
Purpose of the Study:
- To identify the signaling molecules that stimulate carbonic anhydrase synthesis in late-stage chick embryos.
- To elucidate the mechanism of action and regulatory pathways involved in CA induction.
Main Methods:
- In vitro incubation of embryonic erythrocytes with various hormones, second messengers, and embryonic plasma.
- Assay of carbonic anhydrase activity.
- Inhibition studies using actinomycin D, cycloheximide, aluminum fluoride, and pertussis toxin.
- Molecular filtration to determine the size of the active factor.
- Assessment of plasma factor activity under varying oxygen levels.
Main Results:
- N6-phenylisopropyladenosine, an A1 agonist, partially stimulated CA synthesis; adenosine had no effect.
- 10% embryonic plasma from embryos older than 6 days significantly increased CA activity (10-fold in 24 h).
- The plasma-induced increase was blocked by heat inactivation, actinomycin D, cycloheximide, and pertussis toxin, indicating a heat-labile factor acting via transcription and a pertussis toxin-sensitive G protein.
- Mammalian plasma did not affect CA activity, and the active factor's molecular mass is < 2,000 Da.
- In vivo, high blood oxygen partial pressure (Po2) suppresses the plasma factor's action, suggesting Po2-controlled inhibitor synthesis.
Conclusions:
- Embryonic plasma contains a heat-labile factor (< 2,000 Da) that stimulates carbonic anhydrase synthesis through transcriptional activation.
- The factor's receptor is coupled to a pertussis toxin-sensitive G protein.
- In vivo regulation involves suppression of the factor's action by high Po2, likely via an inhibitor molecule.
Abstract:
During chick embryonic development carbonic anhydrase (CA) expression of erythrocytes is kept at a very low level until the last week of incubation (i.e., up to day 14). We have previously obtained evidence that hypoxia is the physiological stimulus for rapid onset of CA synthesis before hatching. Looking for putative signals we have carried out in vitro incubations of embryonic erythrocytes, screening a large number of hormones and second messengers, which were all ineffective, with the exception of the A1 agonist N6-phenylisopropyladenosine (adenosine had no effect). However, incubation with embryonic plasma (10%) from embryos greater than 6 days caused a 10-fold increase of the CA activity during 24 h. This increase was not observed when the incubation was carried out with the addition of actinomycin D, cycloheximide, aluminum fluoride, pertussis toxin, or heat-inactivated plasma. Mammalian plasma had no effect on CA activity. Filtration experiments show that the molecular mass of the factor is less than 2,000 Da. We conclude that embryonic plasma contains a heat-labile factor which stimulates CA synthesis via activation of transcription and whose receptor is coupled to a pertussis toxin-sensitive G protein. In vivo the action of the plasma factor is suppressed as long as blood Po2 is high, suggesting the presence of an inhibitor molecule whose synthesis is controlled by the Po2.