Adenosine causes cAMP-dependent activation of chick embryo red cell carbonic anhydrase and 2,3-DPG synthesis

Abstract

In late chick embryos, coordinate activation of red cell carbonic anhydrase II (CAII) and 2,3-diphosphoglycerate (2,3-DPG) synthesis is initiated by hypoxia. The effects are mediated by unidentified hormonal effectors resident in chick plasma. In the present investigation, we have analyzed the effect of adenosine receptor stimulation on embryonic red cell CAII and 2,3-DPG synthesis. We find that primitive and definitive embryonic red blood cells from chick have an A2a adenosine receptor. Stimulation of the receptor with metabolically stable adenosine analogues causes a large increase of red cell adenosine 3',5'-cyclic monophosphate (cAMP) and subsequent activation of red cell CAII and 2,3-DPG production in definitive red blood cells and of CAII synthesis in primitive red blood cells. Direct stimulation of adenylyl cyclase with forskolin has the same effect. Analysis of red cell protein pattern after labeling with [35S]methionine shows that stimulation of red cell cAMP levels activates synthesis of several other proteins aside from CAII. Presence of actinomycin D inhibits cAMP-dependent changes of protein synthesis, indicating that cAMP-dependent transcriptional activation is required. In contrast to the stable adenosine receptor analogues, adenosine itself was a very weak agonist, unless its metabolism was significantly inhibited. Thus, besides adenosine, other effectors of the adenylyl cyclase system are likely to be involved in the O2 pressure-dependent regulation of red cell metabolism in late development of avian embryos.

cAMP and in vivo hypoxia inducetob,ifr1, andfosexpression in erythroid cells of the chick embryo

“…To confirm the cAMP-dependent expression of the genes identified, we used embryonic RBC of day 11. These cells respond in vivo to hypoxia with induction of 2,3-BPG and CAII synthesis, and in vitro stimulation of these cells with cAMP-elevating agents can simulate these processes in a transcription-dependent manner (11)(12)(13)(14)17). Therefore, the cells are an excellent model to study physiological effects of cAMP on erythroid gene expression.…”

“…In late embryonic development, progressive hypoxia stimulates the release of norepinephrine (NE) and adenosine into the blood (12, 13), which leads to an increase of cAMP in embryonic RBC via ␤-adrenergic and adenosine A 2 receptor activation (13, 17). As a result, the increased intracellular cAMP level activates the coordinate synthesis of carbonic anhydrase (CAII), erythroid 2,3-bisphosphoglycerate (2,3-BPG), the heat shock protein hsp70, and pyrimidine 5Ј-nucleotidase (P5N) in addition to a fall in RBC ATP concentration (11)(12)(13)17). Although the induction of CAII and the change of the RBC organic phosphate pattern are adaptive processes leading to improved O 2 and CO 2 transport of erythroid cells (5), it could be shown that several other RBC proteins are also induced in a cAMP-dependent manner (11), suggesting that cAMP might have additional functions in terminal RBC differentiation.…”

“…We previously showed that immature embryonic RBC (day 3 to day 17) possess a functional cAMP signaling system (3) coupled to ␤-adrenergic and adenosine A 2 receptors. In late embryonic development, progressive hypoxia stimulates the release of norepinephrine (NE) and adenosine into the blood (12,13), which leads to an increase of cAMP in embryonic RBC via ␤-adrenergic and adenosine A 2 receptor activation (13,17). As a result, the increased intracellular cAMP level activates the coordinate synthesis of carbonic anhydrase (CAII), erythroid 2,3-bisphosphoglycerate (2,3-BPG), the heat shock protein hsp70, and pyrimidine 5Ј-nucleotidase (P5N) in addition to a fall in RBC ATP concentration (11)(12)(13)17).…”

Erythroid pyrimidine 5′-nucleotidase: cloning, developmental expression, and regulation by cAMP and in vivo hypoxia

“…11 The 2 hormones that are released by the increasing hypoxia during embryonic development 9,10 also control other processes of late erythroid maturation; they induce regulatory genes (tob, ífr1, fos, hsp70) 12,13 as well as carbonic anhydrase (CAII) and pyrimidine 5Ј-nucleotidase (P5N-I)-2 important erythroid enzymes. 8,9,12,[14][15][16] In humans, the P5N-I enzyme is critically involved in the degradation of pyrimidine nucleotides and ribosomal RNA during final erythroid maturation because an enzyme deficiency causes the accumulation of ribosomal structures and pyrimidine trinucleotides (UTP, CTP). 17,18 Likewise, the chicken embryonic RBCs liberate cellpermeable pyrimidine nucleosides upon hormonal induction of the enzyme, and P5N-I enzyme activity regulates the size of the pyrimidine nucleotide pool.…”

“…It has long been known that in the late chicken embryo, the circulating RBCs adjust their hemoglobin-oxygen affinity to the continuously falling Po 2 by changing their organic phosphate pattern; while in the first two thirds of development, adenosine triphosphate (ATP), uridine triphosphate (UTP), and cytidine triphosphate (CTP) determine the hemoglobin-oxygen affinity, 2,3-bisphosphoglycerate (2,3-BPG) becomes the major organic phosphate of the RBCs in the last week of incubation. [4][5][6][7] The exchange of nucleotides by 2,3-BPG is promoted by the embryonic hormones norepinephrine (NE) and adenosine [8][9][10] and is efficiently blocked by transcriptional inhibition. 11 The 2 hormones that are released by the increasing hypoxia during embryonic development 9,10 also control other processes of late erythroid maturation; they induce regulatory genes (tob, ífr1, fos, hsp70) 12,13 as well as carbonic anhydrase (CAII) and pyrimidine 5Ј-nucleotidase (P5N-I)-2 important erythroid enzymes.…”