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 (DDT1 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 [H-3]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 kappa B (NF-kappa B) activation within 30 min of exposure, a time point that preceded the loss of function of the A(1)AR. Inhibition of NF-kappa B 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-kappa 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-kappa B and [H-3]DPCPX binding. We conclude that pertussis toxin promotes de novo A(1)AR synthesis by activating NF-kappa B through an ADP ribosylation-independent mechanism involving intracellular Ca2+ release and PKC activation.