The A1 adenosine receptor (A1AR) contributes to the cytoprotective action of adenosine under conditions known to generate reactive oxygen species (ROS). Pharmacological manipulation of A1AR expression has been shown to modulate this cytoprotective role. In this study, we provide evidence that ROS generated could increase the expression of the A1AR and thereby offset the detrimental effects of ROS. Incubation of DDT1MF-2 smooth muscle cells with ROS-generating chemotherapeutic agents, such as cisplatin (2.5 microM) or H2O2 (10 microM), elicited an increase in A1AR expression within 24 hr. The induction by H2O2 was reduced by the ROS scavenger catalase but not superoxide dismutase. Inhibition of nuclear factor kappa B (NF kappa B) by pyrrolidine dithiocarbamate (200 microM), dexamethasone (100 nM), or genistein (1 microM) abrogated the cisplatin-mediated increase in A1AR. Cisplatin promoted rapid translocation of NF kappa B (but not AP-1) to the nucleus, as detected by electrophoretic mobility shift assays and by Western blotting. A putative NF kappa B sequence in the A1AR promoter effectively competed with labeled kappa B probe for binding in nuclear preparations derived from DDT1MF-2 cells. Transient transfection of DDT1MF-2 cells with the A1AR promoter coupled to firefly luciferase reporter gene led to cisplatin-inducible and pyrrolidine dithiocarbamate-sensitive luciferase activity, suggesting the presence of functional NF kappa B binding site(s) in the A1AR promoter sequence. Treatment of cells with (R)-phenylisopropyladenosine (1 microM), an agonist of the A1AR, reduced cisplatin-mediated lipid peroxidation, which was reversed after blockade of the A1AR. These data suggest that ROS can increase the expression of the A1AR by activating NF kappa B regulatory site(s) on this gene and thereby enhance the cytoprotective role of adenosine.

Previous studies have indicated that desensitization of the A1 adenosine receptor (A1AR), unlike other adenosine receptor subtypes and G protein-coupled receptors, required prolonged exposure to agonists. We more closely studied this observation by focusing on changes in the A1AR signal transduction pathway after short term agonist exposure (0.5-4 hr) in the hamster vas deferens smooth muscle cell line (DDT1MF-2 cells). Incubation of these cells with 1 microM (R)-phenylisopropyladenosine [(R)-PIA] produced a time-dependent loss in binding of the agonist radioligand [125I]N6-2-(4-amino-3-iodophenyl)ethyladenosine but not of the antagonist radioligand [3H]8-cyclopentyl-1,3-dipropylxanthine. This was accompanied by a reduction in the high affinity (G protein-coupled) state of this receptor from 63 +/- 8% to 37 +/- 12% after treatment for 4 hr. Moreover, cells treated with (R)-PIA demonstrated reduced agonist-stimulated GTPase activity and diminished inhibition of adenylyl cyclase activity but no change in expression of alphai and beta subunits. The decreases in agonist binding in the desensitized cells were reversible after treatment of DDT1MF-2 cell membranes with alkaline phosphatase or protein phosphatases 1 and 2A, suggesting a role of phosphorylation in the uncoupling and desensitization of the A1AR. Incubation of cells with (R)-PIA led to rapid translocation of G protein-coupled receptor kinase (GRK) from the cytosol to the plasma membrane within 1 hr of exposure. In addition, purified preparations of the A1AR that were phosphorylated with purified recombinant GRK-2 demonstrated enhanced affinity for arrestin over Gi/Go. These results indicate rapid and functional desensitization of the A1AR by brief exposure to agonist. The mechanism underlying this event seems to involve phosphorylation of the A1AR, presumably by the GRK or GRKs.

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Nerve growth factor (NGF) induces differentiation of the rat pheochromocytoma clone (PC12) by activating the high affinity receptor, p140(trkA), linked to mitogen-activated protein kinase. While the physiological role of the low affinity NGF receptor (p75) has not been clearly defined, this receptor promotes activation of nuclear factor (NF) kappaB in Schwann cells. PC12 cells express the A(2A) adenosine receptor (AR), whose expression is significantly decreased by NGF treatment. In this study, we determined whether TrkA or p75 is involved in NGF-mediated regulation of A(2A)AR expression. NGF treatment decreased A(2A)AR in a time-dependent manner, with maximal effects observed by 1 day, and continued down-regulation of the receptor for up to 3 days in the presence of NGF. The decrease in A(2A)AR was associated with a more delayed decrease in the steady-state levels of the A(2A)AR mRNA. Down-regulation of the A(2A)AR at 1 day was mimicked by activators of NFkappaB, such as H(2)O(2), and ceramide, and was attenuated by the inhibitor pyrrolidine dithiocarbamate or following transient transfection of PC12 cells with a dominant negative IkappaBalpha mutant. Moreover, NGF stimulated nuclear accumulation of p65 subunits of NFkappaB (but not p50 subunits) in PC12 cells, as determined by electrophoretic mobility shift assays and by Western blotting. In contrast, inhibition of TrkA by AG879 or of TrkA-dependent mitogen-activated protein kinase mitogen-activated protein kinase kinase with PD98059 blocked PC12 cell differentiation without affecting A(2A)AR down-regulation, suggesting dissociation between these two phenomena. Taken together, these data provide strong support for the involvement of the p75/NFkappaB pathway in NGF-mediated down-regulation of A(2A)AR in PC12 cells.