Regulation of Cyclic AMP by the μ‐Opioid Receptor in Human Neuroblastoma SH‐SY5Y Cells

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Abstract
The human neuroblastoma clonal cell line SH-SY5Y expresses both μ- and δ-opioid receptors (ratio ∼4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by μ-opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE,), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (∼65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE, and VIP cAMP response from ∼50 to ∼80%. The use of highly μ- and δ-selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly μ, and not δ, receptor mediated in SH-SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE,-stimulated, RA-differentiated SHSY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over 12h, a fourfold shift of the PGE,-morphine dose-response curve was observed, whether or not IBMX was added. However, μ-opioid receptor number and affinity to the μ-selective [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin were largely unaffected, and Na+- and guanyl nucleotide-induced shifts of morphine-[3H]naloxone competition curves were unchanged. Further, morphine pretreatment elicited an AC rebound effect, causing higher cAMP accumulation after the drug was removed from the medium or acutely antagonized by naloxone. These results document biochemical correlates of μ-opiate tolerance and dependence in SH-SY5Y cells.