ß 1-Adrenergic receptors in human neuroblastoma

Abstract

ß-Adrenergic receptors and adenylate cyclase activity were identified and partially characterized in 10 human neuroblastoma tumors obtained from direct biopsy, from human cell lines grown in the athymic, nude mouse or from established tumor cell lines in tissue culture. Particulate membrane fractions of eight of the tumors contained ß-adrenergic receptor sites identified by the ß-adrenergic antagonists (−)-[ 3H]dihyroalprenolol ((−)-[ 3H]DHA) and (−)-[ 125I]iodohydroxybenzylphindolol ((−)-[ 125I]HYP). Specific binding of high affinity sites (B max) varied greatly among the tumors ranging from non-detectable concentrations to approximately 130 fmol/mg of crude membrane protein. Analysis of the saturation experiments with (−)-[ 3H]DHA resulted in curvilinear Scatchard plots in the tumors, suggesting the presence of multiple classes of sites and there was no evidence of cooperativity in dissociation experiments with (−)-[ 3H]DHA. The high affinity class of sites were inhibited by catecholamines, in contrast to the ineffectiveness of catecholamines in inhibiting (−)-[ 3H]DHA binding at the low affinity class of sites. TheK D of the high affinity site was approximately 1–2 nM for (−)-[ 3H]DHA and was similar in all the tumors studied. A single class of sites was demonstrated with (−)-[ 125I]HYP, in several of the tumors studied,K D approximately 120 pM, and the number of sites determined with (—)-[ 125I]HYP was identical to the number of high affinity sites determined with (−)-[ 3H]DHA. The properties of the receptor were characterized in several tumor lines: SK-N-MC tumor line contained both a ß-adrenergic receptor site and catecholamine sensitive adenylate cyclase activity. Agonists competed for the ß-adrenergic sites in the order of potency (−)-isoproterenol > (−)-norepinephrine = (−)-epinephrine > > (−)-dopamine characteristic of a ß-adrenergic receptor subtype. The affinity of the receptor for the selective agents, metropol (ß 1-selective antagonist) and zinterol (ß 2-selective agonist) was typical of a homogeneous ß 1-adrenergic site. Guanine nucleotides, guanine triphosphate and guanyl-5′-yl-imidophosphate, decreased catecholamine affinity for the receptor site in several tumors studied and enhanced maximal catecholamine stimulated adenylate cyclase activity in the sensitive cell line. Adenylate cyclase activity in particulate fractions of most of the neuroblastoma tumors was responsive to prostaglandins (PGE 1), guanine nucleotide and NaF. While most tumors contained ß-adrenergic receptor sites only SK-N-MC was clearly catecholamine responsive as assessed by catecholamine-sensitive adenylate cyclase activity. Human neuroblastomas, a homogenous tissue of neural crest origin, contained ß-adrenergic receptor sites which varied greatly in number (B max) and in their ability to stimulate adenylate cyclase activity. Guanine nucleotides decreased agonist affinity for the receptor in the lines tested and enhanced prostaglandin and catecholamine sensitive activity in responsive tumor lines.