Induction of adenosine A1 receptor expression by pertussis toxin via an adenosine 5'-diphosphate ribosylation-independent pathway.
Pertussis toxin ADP ribosylates G(i) and G(o) transducing proteins and functionally uncouples adenosine A(1) receptor (A(1)AR) from its effectors. We hypothesized that this loss in receptor coupling could lead to de novo A(1)AR synthesis by the cell in a futile attempt to re-establish normal receptor function. To test this hypothesis, we used hamster ductus deferens tumor (DDT(1) MF-2) cells, a cell culture model for studying A(1)AR, and showed that pertussis toxin (100 ng/ml) produced a time-dependent loss in A(1)AR-G(i) interaction and abolished A(1)AR activation of extracellular signal-regulated kinase 1/2. Interestingly, pertussis toxin increased the expression of A(1)AR, as measured by real-time polymerase chain reaction, immunocytochemistry, and [(3)H]cyclopentyl-1,3-dipropylxanthine (DPCPX) binding, suggesting a compensatory response to G(i) protein inactivation. DDT(1) MF-2 cells exposed to pertussis toxin demonstrated nuclear factor kappaB (NF-kappaB) activation within 30 min of exposure, a time point that preceded the loss of function of the A(1)AR. Inhibition of NF-kappaB attenuated the increase in A(1)AR induced by pertussis toxin. Cells exposed to B-oligomer subunit of pertussis toxin, devoid of significant ADP ribosyltransferase activity, showed increased A(1)AR protein expression, preceded by activation of NF-kappaB. B-Oligomer increased intracellular Ca(2+) in DDT(1) MF-2 cells. Chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or inhibition of protein kinase C (PKC) with bisindolylmaleimide hydrochloride reduced the activation of NF-kappaB and [(3)H]DPCPX binding. We conclude that pertussis toxin promotes de novo A(1)AR synthesis by activating NF-kappaB through an ADP ribosylation-independent mechanism involving intracellular Ca(2+) release and PKC activation.
Jajoo
Sarvesh
S
Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9629, USA.
Mukherjea
Debashree
D
Pingle
Sandeep
S
Sekino
Yuko
Y
Ramkumar
Vickram
V
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D043682:Receptor, Adenosine A1 / Q000096:biosynthesis*; D015398:Signal Transduction / Q000187:drug effects*
D016328:NF-kappa B; D043682:Receptor, Adenosine A1; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium