Activation of cyclic AMP-dependent protein kinase reverses tolerance of a nucleoside transporter to ethanol.
Adenosine mediates some of the acute and chronic effects of ethanol in neural cells. In cultured NG108-15 cells, ethanol inhibits adenosine uptake via a specific facilitative nucleoside transporter leading to an increase in extracellular adenosine, activation of adenosine A2 receptors and increases in intracellular cyclic AMP (cAMP). After chronic ethanol exposure, an adaptive decrease in receptor-stimulated cAMP levels occurs. Additionally, the transporter becomes insensitive to rechallenge with ethanol and adenosine uptake is not inhibited. cAMP levels are decreased in cells chronically exposed to ethanol and we show here that cAMP-dependent kinase (PKA) activity in cellular homogenates also is decreased. Therefore, decreased cAMP-dependent phosphorylation may be responsible for loss of ethanol sensitivity. To test this hypothesis, NG108-15 cells were treated with agents that alter PKA activity and the ethanol sensitivity of adenosine transport was measured. In naive cells, decreasing PKA activity with the cAMP antagonist, Rp-adenosine-3',5'-cyclic phosphorothioate, resulted in ethanol-insensitive adenosine uptake. This effect was blocked by the phosphatase inhibitor, okadaic acid. These results suggest that loss of ethanol sensitivity is correlated with decreased PKA activity. Therefore, stimulating PKA activity in chronically treated cells should restore sensitivity of adenosine uptake to inhibition by ethanol. Indeed, the cAMP agonist, Sp-adenosine-3',5'-cyclic phosphorothioate, restored ethanol sensitivity of transport in cells treated chronically with ethanol. Our results suggest that ethanol sensitivity of adenosine transport is regulated by PKA and protein phosphatase activities in NG108-15 cells. Moreover, the effects of chronic ethanol exposure on adenosine transport can be reversed by activating PKA.
Coe
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Ernest Gallo Clinic and Research Center, University of California, San Francisco, USA.
Dohrman
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DP
Constantinescu
A
A
Diamond
I
I
Gordon
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AS
D000241:Adenosine / Q000378:metabolism; D002352:Carrier Proteins / Q000187:drug effects*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004361:Drug Tolerance; D000431:Ethanol / Q000494:pharmacology*; D008565:Membrane Proteins / Q000187:drug effects*; D033703:Nucleoside Transport Proteins; D010749:Phosphoprotein Phosphatases / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D000431:Ethanol; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D000241:Adenosine