The effects of retinoic acid-induced differentiation on neurotransmitter receptor content and signal transduction in a human neuroblastoma cell line.

摘要

The purpose of the present study was to establish the effects of retinoic acid-induced differentiation on muscarinic receptor populations and signal transduction pathways in the human neuroblastoma Sk-N-SH cells. The human neuroblastoma cell line Sk-N-SH was induced to differentiate by treatment with 1 {dollar}mu{dollar}M retinoic acid for 7 days. Differentiation was characterized by profuse neurite outgrowth, a decrease in cell growth, and a 2-3 fold increase in the protein content of each cell. Muscarinic receptors were labelled using ({dollar}sp3{dollar}H) N-methyl scopolamine. Muscarinic receptor density increased by approximately 36% after treatment for 7 days with retinoic acid (Bmax, control = 126 {dollar}pm{dollar} 13 fmol/mg protein; Bmax, retinoic acid-treated = 170 {dollar}pm{dollar} 17 fmol/mg protein; p {dollar}<{dollar} 0.05), corresponding to a 170% increase in receptor content per cell. The affinity of ({dollar}sp3{dollar}H) NMS for the receptors was somewhat lower in the differentiated cells (K{dollar}sb{lcub}rm D{rcub}{dollar}, control = 0.14 {dollar}pm{dollar} 0.04 nM; K{dollar}sb{lcub}rm D{rcub}{dollar}, retinoic acid-treated = 0.25 {dollar}pm{dollar} 0.04 nM; p {dollar}<{dollar} 0.05). The guanine nucleotide sensitivity of agonist (carbamylcholine) binding to Sk-N-SH muscarinic receptors was slightly decreased by differentiation.; Reverse transcriptase/polymerase chain reaction (PCR) analysis using muscarinic receptor subtype specific primers revealed that undifferentiated Sk-N-SH cells transcribed mRNA for all 5 receptor subtypes; this pattern was not affected by differentiation.; Differentiation did not affect basal G protein GTPase activity. However, acetylcholine (100 {dollar}mu{dollar}M) stimulation of G protein GTPase activity was decreased in differentiated cells (18 {dollar}pm{dollar} 1.8 pmol/min/mg protein) compared to the undifferentiated cells (23 {dollar}pm{dollar} 1.0 pmol/min/mg protein) (p {dollar}<{dollar} 0.05).; Muscarine (0.1-100 {dollar}mu{dollar}M) stimulated {dollar}sp{lcub}45{rcub}{dollar}Ca influx into Sk-N-SH cells, and this uptake was inhibited by preincubation with atropine. The magnitude of the muscarinic receptor-mediated uptake was 50-60% lower in the differentiated cells.; It is demonstrated that in Sk-N-SH cells, retinoic acid-induced differentiation: (1) increases the size of the muscarinic receptor population (Bmax) while decreasing ({dollar}sp3{dollar}H) NMS binding affinity, (2) does not alter muscarinic receptor pharmacology, or the expression of muscarinic receptor subtypes, (3) decreases muscarinic receptor-stimulated {dollar}sp{lcub}45{rcub}{dollar}Ca flux 50-60% compared to undifferentiated cells, (4) depresses basal adenylate cyclase activity, increases fractional stimulation of forskolin-stimulated activity of adenylate cyclase, and may increase muscarinic receptor-mediated inhibition of adenylate cyclase activity, (5) does not alter basal G protein GTPase activity but depresses muscarinic receptor-stimulated high affinity GTPase activity suggesting muscarinic receptor-G protein coupling is altered, and (6) does not alter expression of Go{dollar}sb{lcub}alpha{rcub} rm{lcub}Gs{rcub}sb{lcub}rmalpha{rcub}{dollar} and G{dollar}sb{lcub}beta{rcub}{dollar} content while Gi{dollar}sb{lcub}alpha 3{rcub}{dollar} and Gq{dollar}sb{lcub}alpha{rcub}{dollar} are depressed in differentiated as well as in 0.1% ethanol treated cells. (Abstract shortened by UMI.)