Coe I R, Dohrman D P, Constantinescu A, Diamond I, Gordon A S
Ernest Gallo Clinic and Research Center, University of California, San Francisco, USA.
J Pharmacol Exp Ther. 1996 Feb;276(2):365-9.
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.
腺苷介导乙醇在神经细胞中的一些急性和慢性作用。在培养的NG108 - 15细胞中，乙醇通过特异性易化核苷转运体抑制腺苷摄取，导致细胞外腺苷增加、腺苷A2受体激活以及细胞内环磷酸腺苷（cAMP）增加。长期乙醇暴露后，受体刺激的cAMP水平会适应性降低。此外，转运体对乙醇再次刺激变得不敏感，腺苷摄取不再受到抑制。长期暴露于乙醇的细胞中cAMP水平降低，我们在此表明细胞匀浆中的cAMP依赖性蛋白激酶（PKA）活性也降低。因此，cAMP依赖性磷酸化的降低可能是乙醇敏感性丧失的原因。为了验证这一假设，用改变PKA活性的试剂处理NG108 - 15细胞，并测量腺苷转运的乙醇敏感性。在未处理的细胞中，用cAMP拮抗剂Rp - 腺苷 - 3',5'-环硫代磷酸酯降低PKA活性，导致腺苷摄取对乙醇不敏感。这种作用被磷酸酶抑制剂冈田酸阻断。这些结果表明乙醇敏感性的丧失与PKA活性降低相关。因此，刺激长期处理细胞中的PKA活性应能恢复腺苷摄取对乙醇抑制的敏感性。事实上，cAMP激动剂Sp - 腺苷 - 3',5'-环硫代磷酸酯恢复了长期用乙醇处理的细胞中转运的乙醇敏感性。我们的结果表明，NG108 - 15细胞中腺苷转运的乙醇敏感性受PKA和蛋白磷酸酶活性调节。此外，长期乙醇暴露对腺苷转运的影响可通过激活PKA来逆转。
