A₂B receptors mediate the induction of early genes and inhibition of arterial smooth muscle cell proliferation via Epac.
OBJECTIVE
Extracellular adenosine and adenine nucleotides play important roles in the regulation of the blood vessel tonus and platelet aggregation. Less is known about the effects of these extracellular signalling molecules on gene expression in vascular smooth muscle cells involved in long-term vascular effects. In the present study, we therefore searched for adenosine-induced changes in the expression of early genes in cultured human coronary artery smooth muscle cells (HCASMCs).


RESULTS
Whole-genome DNA array hybridization revealed that adenosine induced a set of early genes including the nuclear receptor subfamily 4, group A, member 1 (NR4A1/Nur77/TR3). The pattern of the effects of adenosine on gene expression resembles the change in expression induced by the direct activator of adenylate cyclase forskolin. Real-time reverse-transcriptase PCR confirmed that adenosine and its analogue N-ethyl-carboxamidoadenosine elicited a strong induction of NR4A1. These effects were markedly attenuated by A(2B) receptor antagonists including 8-[4-(4-benzylpiperazide-1-sulfonyl)phenyl]-1-propylxanthine (PSB-601) and were mimicked by a cyclic AMP (cAMP) analogue [8-(4-chlorophenylthio)-2'-O-methyl-cAMP, 8CPT] acting on the exchange protein activated by cAMP (Epac). Long-term experiments over 5 days showed that 2-chloroadenosine decreased cell proliferation in the presence of platelet-derived growth factor. This effect of 2-chloroadenosine was also attenuated by PSB-601 and mimicked by 8CPT. Treatment with small interfering RNA directed against NR4A1 attenuated the inhibitory effect of 8CPT on proliferation.


CONCLUSIONS
In summary, our results demonstrate the operation of adenosine A₂(B) receptors mediating an early induction of NR4A1 and a decrease in cell proliferation via the cAMP/Epac pathway in HCASMCs.
Federal Institute for Drugs and Medical Devices, Bonn, Germany. p.mayer@bfarm.de
Peter
P
Mayer
Annette Viktoria
AV
Hinze
Anja
A
Harst
Ivar
I
von Kügelgen
D000241:Adenosine / Q000378:metabolism*; D058908:Adenosine A2 Receptor Agonists / Q000494:pharmacology; D058917:Adenosine A2 Receptor Antagonists / Q000494:pharmacology; D000328:Adult; D000368:Aged; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D043704:Receptor, Adenosine A2B / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
D058908:Adenosine A2 Receptor Agonists; D058917:Adenosine A2 Receptor Antagonists; D020662:Guanine Nucleotide Exchange Factors; C539821:NR4A1 protein, human; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; C116122:RAPGEF3 protein, human; D043704:Receptor, Adenosine A2B; D000242:Cyclic AMP; D000241:Adenosine