Ca2+-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine

U. K. Schubart, N. Fleischer, J. Erlichman

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

Ca2+-dependent protein phosphorylation was studied in intact hamster insulinoma cells. Depolarizing concentrations of potassium which stimulate Ca2+ uptake and insulin release by these cells also increased phosphorylation of one peptide, M(r)=60,000 (P60). This was demonstrated by incubating 32P-labeled insulinoma cells in media containing 50 mM K+ followed by analysis of the cellular proteins by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography. Potassium-induced phosphorylation of P60 was nearly half-maximal after 1 min and reached a plateau by 10 min. The enhanced 32P-labeling of P60 observed in the presence of 50mM K+ was Ca2+-dependent since omission of extracellular Ca2+ or addition of the Ca2+ channel blocker α-isopropyl-α-[(N-methyl-N-homoveratryl)-γ-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 μM), which stimulates insulin release in a cAMP-dependent manner, had no effect on P60 phosphorylation. A possible involvement of calmodulin was explored in studies using trifluoperazine. The Ca2+ dependent increase in phosphorylation of P60 was prevented by trifluoperazine. Moreover, Ca2+ influx-mediated insulin release and P60 phosphorylation were inhibited at nearly identical concentrations of trifluoperazine. Half-maximal inhibition of potassium-induced insulin release and P60 phosphorylation was seen at 2.6 μM and 2.5 μM trifluoperazine, respectively. The data are consistent with a sequence of events involving Ca2+ influx, phosphorylation of P60 by a calmodulin-dependent protein kinase, and resultant insulin secretion.

Original languageEnglish (US)
Pages (from-to)11063-11066
Number of pages4
JournalJournal of Biological Chemistry
Volume255
Issue number23
StatePublished - 1980

Fingerprint

Trifluoperazine
Insulinoma
Phosphorylation
Cricetinae
Insulin
Proteins
Potassium
Calcium-Calmodulin-Dependent Protein Kinases
Calmodulin
Electrophoresis
Glucagon
Autoradiography
Sodium Dodecyl Sulfate
Labeling
Polyacrylamide Gel Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ca2+-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine. / Schubart, U. K.; Fleischer, N.; Erlichman, J.

In: Journal of Biological Chemistry, Vol. 255, No. 23, 1980, p. 11063-11066.

Research output: Contribution to journalArticle

@article{fc18a52d8dc64d439d8a80975999bdce,
title = "Ca2+-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine",
abstract = "Ca2+-dependent protein phosphorylation was studied in intact hamster insulinoma cells. Depolarizing concentrations of potassium which stimulate Ca2+ uptake and insulin release by these cells also increased phosphorylation of one peptide, M(r)=60,000 (P60). This was demonstrated by incubating 32P-labeled insulinoma cells in media containing 50 mM K+ followed by analysis of the cellular proteins by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography. Potassium-induced phosphorylation of P60 was nearly half-maximal after 1 min and reached a plateau by 10 min. The enhanced 32P-labeling of P60 observed in the presence of 50mM K+ was Ca2+-dependent since omission of extracellular Ca2+ or addition of the Ca2+ channel blocker α-isopropyl-α-[(N-methyl-N-homoveratryl)-γ-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 μM), which stimulates insulin release in a cAMP-dependent manner, had no effect on P60 phosphorylation. A possible involvement of calmodulin was explored in studies using trifluoperazine. The Ca2+ dependent increase in phosphorylation of P60 was prevented by trifluoperazine. Moreover, Ca2+ influx-mediated insulin release and P60 phosphorylation were inhibited at nearly identical concentrations of trifluoperazine. Half-maximal inhibition of potassium-induced insulin release and P60 phosphorylation was seen at 2.6 μM and 2.5 μM trifluoperazine, respectively. The data are consistent with a sequence of events involving Ca2+ influx, phosphorylation of P60 by a calmodulin-dependent protein kinase, and resultant insulin secretion.",
author = "Schubart, {U. K.} and N. Fleischer and J. Erlichman",
year = "1980",
language = "English (US)",
volume = "255",
pages = "11063--11066",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "23",

}

TY - JOUR

T1 - Ca2+-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine

AU - Schubart, U. K.

AU - Fleischer, N.

AU - Erlichman, J.

PY - 1980

Y1 - 1980

N2 - Ca2+-dependent protein phosphorylation was studied in intact hamster insulinoma cells. Depolarizing concentrations of potassium which stimulate Ca2+ uptake and insulin release by these cells also increased phosphorylation of one peptide, M(r)=60,000 (P60). This was demonstrated by incubating 32P-labeled insulinoma cells in media containing 50 mM K+ followed by analysis of the cellular proteins by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography. Potassium-induced phosphorylation of P60 was nearly half-maximal after 1 min and reached a plateau by 10 min. The enhanced 32P-labeling of P60 observed in the presence of 50mM K+ was Ca2+-dependent since omission of extracellular Ca2+ or addition of the Ca2+ channel blocker α-isopropyl-α-[(N-methyl-N-homoveratryl)-γ-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 μM), which stimulates insulin release in a cAMP-dependent manner, had no effect on P60 phosphorylation. A possible involvement of calmodulin was explored in studies using trifluoperazine. The Ca2+ dependent increase in phosphorylation of P60 was prevented by trifluoperazine. Moreover, Ca2+ influx-mediated insulin release and P60 phosphorylation were inhibited at nearly identical concentrations of trifluoperazine. Half-maximal inhibition of potassium-induced insulin release and P60 phosphorylation was seen at 2.6 μM and 2.5 μM trifluoperazine, respectively. The data are consistent with a sequence of events involving Ca2+ influx, phosphorylation of P60 by a calmodulin-dependent protein kinase, and resultant insulin secretion.

AB - Ca2+-dependent protein phosphorylation was studied in intact hamster insulinoma cells. Depolarizing concentrations of potassium which stimulate Ca2+ uptake and insulin release by these cells also increased phosphorylation of one peptide, M(r)=60,000 (P60). This was demonstrated by incubating 32P-labeled insulinoma cells in media containing 50 mM K+ followed by analysis of the cellular proteins by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography. Potassium-induced phosphorylation of P60 was nearly half-maximal after 1 min and reached a plateau by 10 min. The enhanced 32P-labeling of P60 observed in the presence of 50mM K+ was Ca2+-dependent since omission of extracellular Ca2+ or addition of the Ca2+ channel blocker α-isopropyl-α-[(N-methyl-N-homoveratryl)-γ-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 μM), which stimulates insulin release in a cAMP-dependent manner, had no effect on P60 phosphorylation. A possible involvement of calmodulin was explored in studies using trifluoperazine. The Ca2+ dependent increase in phosphorylation of P60 was prevented by trifluoperazine. Moreover, Ca2+ influx-mediated insulin release and P60 phosphorylation were inhibited at nearly identical concentrations of trifluoperazine. Half-maximal inhibition of potassium-induced insulin release and P60 phosphorylation was seen at 2.6 μM and 2.5 μM trifluoperazine, respectively. The data are consistent with a sequence of events involving Ca2+ influx, phosphorylation of P60 by a calmodulin-dependent protein kinase, and resultant insulin secretion.

UR - http://www.scopus.com/inward/record.url?scp=0019231295&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0019231295&partnerID=8YFLogxK

M3 - Article

C2 - 6254954

AN - SCOPUS:0019231295

VL - 255

SP - 11063

EP - 11066

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 23

ER -