Alpha 2-adrenergic and muscarinic cholinergic receptors have opposing actions on cyclic AMP levels in SK-N-SH human neuroblastoma cells.
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 microM, 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, muscarinic 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 microM, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Baron
B M
BM
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Siegel
B W
BW
D037361:Adenylate Cyclase Toxin; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism*; D037342:Pertussis Toxin; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D011942:Receptors, Adrenergic, alpha; D011964:Receptors, Gastrointestinal Hormone; D011976:Receptors, Muscarinic; D018005:Receptors, Vasoactive Intestinal Peptide; D010566:Virulence Factors, Bordetella; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP; D037342:Pertussis Toxin