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Article: Intracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosis

TitleIntracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosis
Authors
Issue Date2014
Citation
Biochemical and Biophysical Research Communications, 2014, v. 450, p. 93-98 How to Cite?
AbstractPrevious studies suggested indirectly that vascular endothelial cells (VECs) might be able to release intracellularly-formed adenosine. We isolated VECs from the rat soleus muscle using collagenase digestion and magnetic-activated cell sorting (MACS). The VEC preparation had >90% purity based on cell morphology, fluorescence immunostaining, and RT-PCR of endothelial markers. The kinetic properties of endothelial cytosolic 5’-nucleotidase suggested it was the AMP-preferring N-I isoform: its catalytic activity was 4 times higher than ecto-5’nucleotidase. Adenosine kinase had 50 times greater catalytic activity than adenosine deaminase, suggesting that adenosine removal in VECs is mainly through incorporation into adenine nucleotides. The maximal activities of cytosolic 5’-nucleotidase and adenosine kinase were similar. Adenosine and ATP accumulated in the medium surrounding VECs in primary culture. Hypoxia doubled the adenosine, but ATP was unchanged; AOPCP did not alter medium adenosine, suggesting that hypoxic VECs had released intracellularly-formed adenosine. Acidosis increased medium ATP, but extracellular conversion of ATP to AMP was inhibited, and adenosine remained unchanged. Acidosis in the buffer-perfused rat gracilis muscle elevated AMP and adenosine in the venous effluent, but AOPCP abolished the increase in adenosine, suggesting that adenosine is formed extracellularly by non-endothelial tissues during acidosis in vivo. Hypoxia plus acidosis increased medium ATP by a similar amount to acidosis alone and adenosine 6-fold; AOPCP returned the medium adenosine to the level seen with hypoxia alone. These data suggest that VECs release intracellularly formed adenosine in hypoxia, ATP during acidosis, and both under simulated ischaemic conditions, with further extracellular conversion of ATP to adenosine.
Persistent Identifierhttp://hdl.handle.net/10722/203339

 

DC FieldValueLanguage
dc.contributor.authorLe, GYen_US
dc.contributor.authorEssackjee, HCen_US
dc.contributor.authorBallard, HJen_US
dc.date.accessioned2014-09-19T14:09:00Z-
dc.date.available2014-09-19T14:09:00Z-
dc.date.issued2014en_US
dc.identifier.citationBiochemical and Biophysical Research Communications, 2014, v. 450, p. 93-98en_US
dc.identifier.urihttp://hdl.handle.net/10722/203339-
dc.description.abstractPrevious studies suggested indirectly that vascular endothelial cells (VECs) might be able to release intracellularly-formed adenosine. We isolated VECs from the rat soleus muscle using collagenase digestion and magnetic-activated cell sorting (MACS). The VEC preparation had >90% purity based on cell morphology, fluorescence immunostaining, and RT-PCR of endothelial markers. The kinetic properties of endothelial cytosolic 5’-nucleotidase suggested it was the AMP-preferring N-I isoform: its catalytic activity was 4 times higher than ecto-5’nucleotidase. Adenosine kinase had 50 times greater catalytic activity than adenosine deaminase, suggesting that adenosine removal in VECs is mainly through incorporation into adenine nucleotides. The maximal activities of cytosolic 5’-nucleotidase and adenosine kinase were similar. Adenosine and ATP accumulated in the medium surrounding VECs in primary culture. Hypoxia doubled the adenosine, but ATP was unchanged; AOPCP did not alter medium adenosine, suggesting that hypoxic VECs had released intracellularly-formed adenosine. Acidosis increased medium ATP, but extracellular conversion of ATP to AMP was inhibited, and adenosine remained unchanged. Acidosis in the buffer-perfused rat gracilis muscle elevated AMP and adenosine in the venous effluent, but AOPCP abolished the increase in adenosine, suggesting that adenosine is formed extracellularly by non-endothelial tissues during acidosis in vivo. Hypoxia plus acidosis increased medium ATP by a similar amount to acidosis alone and adenosine 6-fold; AOPCP returned the medium adenosine to the level seen with hypoxia alone. These data suggest that VECs release intracellularly formed adenosine in hypoxia, ATP during acidosis, and both under simulated ischaemic conditions, with further extracellular conversion of ATP to adenosine.en_US
dc.languageengen_US
dc.relation.ispartofBiochemical and Biophysical Research Communicationsen_US
dc.titleIntracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosisen_US
dc.typeArticleen_US
dc.identifier.emailBallard, HJ: ballard@hkucc.hku.hken_US
dc.identifier.authorityBallard, HJ=rp00367en_US
dc.identifier.doi10.1016/j.bbrc.2014.05.066en_US
dc.identifier.hkuros240434en_US
dc.identifier.volume450en_US
dc.identifier.spage93en_US
dc.identifier.epage98en_US

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