enkephalin--leucine-2-alanine and Neoplasms--Nerve-Tissue

Monolayer cultures of neuroblastoma X glioma hybrid (clonal) cell line NG108-15, synchronized by the isoleucine/glutamine deprivation method, showed maximal expression of opiate binding sites at the same point in the cell cycle at which prostaglandin E1 (PGE1) had a maximum stimulatory effect on cyclic AMP synthesis. However, the capacity of enkephalin [D-Ala2D-Leu5] to block the stimulation of cyclic AMP synthesis by PGE1 was not related to the number of opiate receptors expressed. The Ki for the inhibition of cyclic AMP synthesis by opioid peptides increased substantially during the period of the cell cycle at which maximal expression of opiate binding sites occurred, making the effective level of inhibition of adenylate cyclase activity by 0.1 microM enkephalin [D-Ala2D-Leu5] the same through the cell cycle. Data are presented to suggest that enkephalin receptor coupling to adenylate cyclase, via a GTP-binding protein, is maximal during G1 phase (which may approximate the state of the differentiated neuron) and minimal during S + G2 phase, just prior to cell division, when many receptors are uncoupled.

Topics: Adenylyl Cyclases; Alprostadil; Animals; Cell Cycle; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Guanylyl Imidodiphosphate; Hybrid Cells; Mice; Neoplasms, Nerve Tissue; Prostaglandins E; Rats; Receptors, Opioid

Electron inactivation analysis revealed that the opiate (enkephalin) binding site in neurotumor cell lines NG108-15 and NCB-20 had an apparent target size of 200,000 daltons. Expression of functional opiate receptors in neurotumor cells appeared to require glycosylation, as treatment of such cells with tunicamycin (TM; under conditions where de novo glycosylation of asparagine residues in protein was reduced by 80%, but overall protein and DNA synthesis were inhibited by less than 10%) resulted in the loss of 50% of the opiate binding sites. The loss of binding sites could not be prevented by addition of protease inhibitors to cell cultures, but binding sites were partially restored 48-60 h after removal of the TM. In addition, the number of enkephalin binding sites in TM-treated cells was also restored to near-normal levels by addition of physiological concentrations (1-10 mM) of manganese ions to the in vitro receptor binding incubation mixture. TM treatment resulted in receptor supersensitivity to manganese ions for both opiate agonists and antagonists, no change in the sodium effect for either agonists or antagonists, and subsensitivity to GTP for both agonists and antagonists. However, opiate binding to cell membranes was not substantially inhibited by either neuraminidase treatment or short-term incubation with lectins such as wheat germ agglutinin, ricin, or concanavalin A. Thus, the data suggest that oligosaccharide units are not directly involved in opiate receptor-ligand interactions, but protein glycosylation is required for functional expression of receptors.

Topics: Animals; Carbohydrate Metabolism; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Mice; Neoplasms, Nerve Tissue; Rats; Receptors, Opioid; Tunicamycin