α2‐Adrenergic and Muscarinic Cholinergic Receptors Have Oposing Actions on Cyclic AMP Levels in SK‐N‐SH Human Neuroblastoma Cells

Abstract

Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK-N-SH cells. The .alpha.2-adrenergic agonist UK 14304 decreased forskolin-stimulated cyclic AMP levels by 40 .+-. 2%, with an EC50 of 83 .+-. 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50 = 1 ng/ml) or by the .alpha.2-antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 .+-. 0.1, 2.2 .+-. 0.3, and 1.4 .+-. 0.1 .mu.M, respectively. This finding suggests the presence of normal inhibitory coupling of SK-N-SH cell .alpha.2-adrenergic receptors to adenylate cyclase via the inhibitory GTP-binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK-N-SH cells, msucarinic agonists synergistically increased (67-95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremorine facilitation of the forskolin response were 1.2 .+-. 0.2 and 0.3 .+-. 0.1 .mu.M, respectively. Pharmacological studies using the muscarinic receptor subtype-preferring antagonists 4-diphenylacetoxy-N-methylpiperidine, pirenzepine, and AF-DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 .+-. 1, 16,857 .+-. 757, and 148,043 .+-. 16,209 nM, respectively. CCh-elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK-N-SH cell membranes. Our results indicated the presence of a single CCh binding site with an apparent affinity (Ki = 5 .+-. 1 .mu.M) consistent with that concentration of agonist required to evoke biochemical responses in the intact cell.