Identification of Multiple Phosphoinositide-Linked Receptors on Human SK-N-MC Neuroepithelioma Cells

Abstract

:The biochemical and pharmacological characteristics of receptor-stimulated phosphoinositide (PPI) hydrolysis in human SK-N-MC neuroepithelioma cells have been examined. Of 11 ligands tested, the addition of four, i.e., nor-epinephrine, oxotremorine-M, endothelin-1, and ATP, each resulted in an increased release (three-to eightfold) of inositol phosphates from [ 3 H]inositol-prelabeled cells. Agonist-stimulated PPI turnover was sustained for at least 30 min and required the addition of Ca 2+ for full effect. An increased release of inositol phosphates could also be elicited by the addition of the Ca 2+ ionophore, ionomycin. All four agonists enhanced the release of radiolabeled inositol mono-and bis-phosphates, inositol 1,3,4-trisphosphate, and inositol tetra-kisphosphate. Increases in inositol 1,4,5-trisphosphate were smaller and only consistently observed in the presence of norepinephrine or oxotremorine-M. Norepinephrine-stim-ulated PPI turnover was potently inhibited by prazosin, WB-4101, and 5-methylurapidil ( K 1 , < 2.5 n M ), but was relatively insensitive to chloroethylclonidine pretreatment. This pharmacological profile is consistent with the involvement of an Α 1A -receptor subtype. The presence of an M 1 muscannic cholinergic receptor is also indicated, because pirenzepine blocked oxotremorine-M-stimulated inositol phosphate release ( K 1 = 35 n M ) with a 30-fold greater potency than the M 2 -selective antagonist, AF-DX 116. Of the three endothelins tested, only the addition of endothelin-1 and endothelin-2 promoted PPI hydrolysis, whereas endothelin-3 was essentially inactive. A P 2 nucleotide receptor of broad agonist specificity is also present on these cells and activates PPI turnover in the absence of a generalized increase in plasma membrane permeability. These results indicate that SK-N-MC cells express at least four PPI-linked receptors. Because the functional coupling of three of these receptors, i.e., Α 1A -adrenergic, endothelin, and P 2 nucleotide, has not been extensively characterized previously in neural tissues, the SK-N-MC cell line may provide a useful model system for studies of these receptors and their regulation.