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CELLULAR AND MOLECULAR

Induction of Adenosine A₁ Receptor Expression by Pertussis Toxin via an Adenosine 5'-Diphosphate Ribosylation-Independent Pathway

Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois (S.J., D.M., V.R.); Department of Pharmacology, Georgetown University, Washington, DC (S.P.); and Gunma University School of Medicine, Maebashi, Japan (Y.S.)

Pertussis toxin ADP ribosylates G_i and G_o transducing proteins and functionally uncouples adenosine A₁ receptor (A₁AR) from its effectors. We hypothesized that this loss in receptor coupling could lead to de novo A₁AR synthesis by the cell in a futile attempt to re-establish normal receptor function. To test this hypothesis, we used hamster ductus deferens tumor (DDT₁ MF-2) cells, a cell culture model for studying A₁AR, and showed that pertussis toxin (100 ng/ml) produced a time-dependent loss in A₁AR-G_i interaction and abolished A₁AR activation of extracellular signal-regulated kinase 1/2. Interestingly, pertussis toxin increased the expression of A₁AR, as measured by real-time polymerase chain reaction, immunocytochemistry, and [³H]cyclopentyl-1,3-dipropylxanthine (DPCPX) binding, suggesting a compensatory response to G_i protein inactivation. DDT₁ MF-2 cells exposed to pertussis toxin demonstrated nuclear factor B (NF-B) activation within 30 min of exposure, a time point that preceded the loss of function of the A₁AR. Inhibition of NF-B attenuated the increase in A₁AR induced by pertussis toxin. Cells exposed to B-oligomer subunit of pertussis toxin, devoid of significant ADP ribosyltransferase activity, showed increased A₁AR protein expression, preceded by activation of NF-B. B-Oligomer increased intracellular Ca²⁺ in DDT₁ MF-2 cells. Chelation of intracellular Ca²⁺ 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-B and [³H]DPCPX binding. We conclude that pertussis toxin promotes de novo A₁AR synthesis by activating NF-B through an ADP ribosylation-independent mechanism involving intracellular Ca²⁺ release and PKC activation.

Address correspondence to: Dr. Vickram Ramkumar, Southern Illinois University School of Medicine, P.O. Box 19629, Springfield, IL 62794-9629. E-mail: vramkumar{at}siumed.edu

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