Abstract
We have shown that ethanol inhibits uptake of adenosine by a specific nucleoside transporter in NG108-15 neuroblastoma x glioma cells and that cAMP-dependent protein kinase (PKA) activity is required for this inhibition. After chronic exposure to ethanol, adenosine uptake is no longer inhibited on rechallenge with ethanol, i.e. transport has become tolerant to ethanol. Here we show that protein kinase C (PKC) contributes to ethanol-induced tolerance of adenosine transport. Activation of PKC by phorbol esters in control cells results in an ethanol-tolerant phenotype, similar to that produced by chronic ethanol exposure. In addition, chronic exposure to ethanol increases the amounts of alpha, delta, and epsilon PKC. However, reducing PKC activity by inhibition with chelerythrine during chronic exposure to ethanol or down-regulation by phorbol esters prevents the development of ethanol-induced tolerance of adenosine transport. By contrast, the inhibition of PKA activity produces tolerance to ethanol inhibition of adenosine uptake. When protein phosphatase inhibitors are present, inhibiting PKA activity has no effect on ethanol sensitivity of adenosine uptake, suggesting a role for protein phosphatases in the regulation of ethanol sensitivity of uptake. Taken together, our results suggest that PKA and PKC have opposing effects on the ethanol sensitivity of adenosine transport; PKA activity is required for ethanol sensitivity, and PKC activation produces tolerance. Based on these data, we propose that chronic ethanol exposure increases PKC activity, leading to the activation of a protein phosphatase (1 or 2A). This phosphatase then dephosphorylates a PKA-phosphorylated site, which is required for ethanol to inhibit adenosine uptake. Therefore, the sensitivity of adenosine transport to ethanol appears to be maintained by a balance of PKA and protein phosphatase activities, and PKC may regulate phosphatase activity.
Highlights
We have presented evidence that the cAMP signaling system is important for regulating the response to ethanol in a cell culture model of tolerance
This loss of ethanol sensitivity of adenosine transport was similar to the tolerance caused by chronic ethanol exposure (Fig. 1). -phorbol 12-myristate 13-acetate (PMA) had no further effect on the tolerance of adenosine uptake in cells treated chronically with ethanol (Fig. 1)
The inactive phorbol ester ␣-PMA had no effect on the ethanol sensitivity of adenosine uptake in control cells (Fig. 1), suggesting that the ethanol tolerance produced by -PMA was due to the activation of protein kinase C (PKC)
Summary
From the ‡Ernest Gallo Clinic and Research Center and the ¶Department of Neurology, ʈDepartment of Cellular and Molecular Pharmacology and **Neuroscience Program, University of California, San Francisco, California 94110. Our results suggest that PKA and PKC have opposing effects on the ethanol sensitivity of adenosine transport; PKA activity is required for ethanol sensitivity, and PKC activation produces tolerance Based on these data, we propose that chronic ethanol exposure increases PKC activity, leading to the activation of a protein phosphatase (1 or 2A). We propose that chronic ethanol exposure increases PKC activity, leading to the activation of a protein phosphatase (1 or 2A) This phosphatase dephosphorylates a PKA-phosphorylated site, which is required for ethanol to inhibit adenosine uptake. The inhibition of PKA in naive cells reproduces the ethanol-tolerant phenotype This latter effect can be prevented by inhibiting protein phosphatase activity with okadaic acid (4), suggesting that protein phosphatase (1 or 2A) is involved in regulating the ethanol sensitivity of adenosine uptake. Our results suggest that PKC activity is required for the development of tolerance of adenosine transport after chronic exposure to ethanol
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