The Effects of Retinoic Acid-Induced Differentiation on Neurotransmitter Receptor Content and Signal Transduction in a Human Neuroblastoma Cell Line

Hdl Handle:
http://hdl.handle.net/10675.2/344208
Title:
The Effects of Retinoic Acid-Induced Differentiation on Neurotransmitter Receptor Content and Signal Transduction in a Human Neuroblastoma Cell Line
Authors:
Baumgartner, Melissa K.
Abstract:
The purpose of the present study was to establish the effects of retinoic acidinduced 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 jiM 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 [^H]Nmethyl scopolamine. Muscarinic receptor density increased by approximately 36% after treatment for 7 days with retinoic acid (Bmax, control = 126 ± 13fm ol/m g protein; Bmax, retinoic acid-treated = 170 ± 17 fm ol/m g protein; p<0.05), corresponding to a 170% increase in receptor content per cell. The affinity of I^HJNMS fo r the receptors was somewhat lower in the differentiated cells (Kp, control = 0.14 ± 0.04 nM; Kp, retinoic acid-treated = 0.25 + 0.04 nM; p<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 undifferentiatied Sk- N-SH cells transcribed mRNA for all 5 receptor subtypes; this pattern was not affected by differentiation. [^HJNMS displacement curves with subtype-selective receptor ligands (pirenzepine, m l; AFDX-116, m2; 4-DAMP, m3) indicated the predominant expression of m l and m3 receptor subtypes, and differentiation did Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. not affect the pharmacological profile of the expressed muscarinic receptor populations. Differentiation did not affect basal G protein GTPase activity. However, acetylcholine (100 jiM ) stimulation of G protein GTPase activity was decreased in differentiated cells (18 ± 1.8 pm ol/m in/m g protein) compared to the undifferentiatied cells (23 ± 1.0 pm o l/m in /m g protein) (p<0.05). Inhibition of acetylcholine-stimulated GTPase activity with selective muscarinic receptor antagonists indicated that the m3 antagonist (4-DAMP) was as effective as atropine in inhibiting activity by 80-100%. Selective m l and m2 antagonists were less effective (30-40%) at inhibiting stimulated GTPase activity. There were no differences in inhibition of stimulated GTPase activity after differentiation. Immunoblots of control and retinoic acid-treated cells revealed no change in Goo, Gsa or Gp content after differentiation, however 0.1% ethanol and retinoic acidtreated cells displayed a 30% decrease in expression of Gi<x3, and Gqa. Muscarine (0.1-100 p.M) stimulated 45Ca 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 differentiatied cells. Basal adenylate cyclase activity was depressed in the differentiated cells (2.5 pm ol/ m in/m g protein) compared to the undifferentiated cells (8.4 p m o l/m in /m g protein) (p< 0.05). Forskolin (5 - 50 p,M)-stimulated adenylate cyclase activity was not altered, however fractional stimulation was significantly (p<0.0001) increased in the differentiated cells. Differentiated cells displayed a slightly greater receptor-mediated inhibition of the adenylate cyclase activity by carbamylcholine(1 JiM - I m M ) . 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 [3H]NMS binding affinity, 2) does not alter muscarinic receptor pharmacology, or the expression of muscarinic receptor subtypes, 3) decreases muscarinic receptor-stimulated 45Ca 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<x, Gsa and Gp content while Gfca and Gq<* are depressed in differentiated as well as in 0.1% ethanol treated cells.
Affiliation:
Department of Pharmacology and Toxicology
Issue Date:
Jan-1993
URI:
http://hdl.handle.net/10675.2/344208
Additional Links:
http://ezproxy.augusta.edu/login?url=http://search.proquest.com/docview/304072934?accountid=12365
Type:
Dissertation
Appears in Collections:
Department of Pharmacology and Toxicology Theses and Dissertations; Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorBaumgartner, Melissa K.en
dc.date.accessioned2015-02-05T01:52:29Z-
dc.date.available2015-02-05T01:52:29Z-
dc.date.issued1993-01-
dc.identifier.urihttp://hdl.handle.net/10675.2/344208-
dc.description.abstractThe purpose of the present study was to establish the effects of retinoic acidinduced 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 jiM 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 [^H]Nmethyl scopolamine. Muscarinic receptor density increased by approximately 36% after treatment for 7 days with retinoic acid (Bmax, control = 126 ± 13fm ol/m g protein; Bmax, retinoic acid-treated = 170 ± 17 fm ol/m g protein; p<0.05), corresponding to a 170% increase in receptor content per cell. The affinity of I^HJNMS fo r the receptors was somewhat lower in the differentiated cells (Kp, control = 0.14 ± 0.04 nM; Kp, retinoic acid-treated = 0.25 + 0.04 nM; p<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 undifferentiatied Sk- N-SH cells transcribed mRNA for all 5 receptor subtypes; this pattern was not affected by differentiation. [^HJNMS displacement curves with subtype-selective receptor ligands (pirenzepine, m l; AFDX-116, m2; 4-DAMP, m3) indicated the predominant expression of m l and m3 receptor subtypes, and differentiation did Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. not affect the pharmacological profile of the expressed muscarinic receptor populations. Differentiation did not affect basal G protein GTPase activity. However, acetylcholine (100 jiM ) stimulation of G protein GTPase activity was decreased in differentiated cells (18 ± 1.8 pm ol/m in/m g protein) compared to the undifferentiatied cells (23 ± 1.0 pm o l/m in /m g protein) (p<0.05). Inhibition of acetylcholine-stimulated GTPase activity with selective muscarinic receptor antagonists indicated that the m3 antagonist (4-DAMP) was as effective as atropine in inhibiting activity by 80-100%. Selective m l and m2 antagonists were less effective (30-40%) at inhibiting stimulated GTPase activity. There were no differences in inhibition of stimulated GTPase activity after differentiation. Immunoblots of control and retinoic acid-treated cells revealed no change in Goo, Gsa or Gp content after differentiation, however 0.1% ethanol and retinoic acidtreated cells displayed a 30% decrease in expression of Gi<x3, and Gqa. Muscarine (0.1-100 p.M) stimulated 45Ca 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 differentiatied cells. Basal adenylate cyclase activity was depressed in the differentiated cells (2.5 pm ol/ m in/m g protein) compared to the undifferentiated cells (8.4 p m o l/m in /m g protein) (p< 0.05). Forskolin (5 - 50 p,M)-stimulated adenylate cyclase activity was not altered, however fractional stimulation was significantly (p<0.0001) increased in the differentiated cells. Differentiated cells displayed a slightly greater receptor-mediated inhibition of the adenylate cyclase activity by carbamylcholine(1 JiM - I m M ) . 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 [3H]NMS binding affinity, 2) does not alter muscarinic receptor pharmacology, or the expression of muscarinic receptor subtypes, 3) decreases muscarinic receptor-stimulated 45Ca 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<x, Gsa and Gp content while Gfca and Gq<* are depressed in differentiated as well as in 0.1% ethanol treated cells.en
dc.relation.urlhttp://ezproxy.augusta.edu/login?url=http://search.proquest.com/docview/304072934?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectHuman neuroblastomaen
dc.subjectMuscarinic receptorsen
dc.subjectG proteinsen
dc.subjectDifferentiationen
dc.subjectRetinoic aciden
dc.subjectAdenylate cyclaseen
dc.titleThe Effects of Retinoic Acid-Induced Differentiation on Neurotransmitter Receptor Content and Signal Transduction in a Human Neuroblastoma Cell Lineen
dc.typeDissertationen
dc.contributor.departmentDepartment of Pharmacology and Toxicologyen
dc.description.advisorAronstam, Robert S.en
dc.description.committeeIkeda, Stephen; Lewis, Deborah; Creazzo, Tony; Daniell, Lauraen
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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