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Title: Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, Ca sup 2+ , and protein kinase C

Abstract

Vasoactive intestinal peptide synergistically stimulated initiation of DNA synthesis in Swiss 3T3 cells. The peptide stimulated ({sup 3}H)thymidine incorporation in the presence of insulin and either forskolin or an inhibitor of cAMP phosphodiesterase in a concentration-dependent manner. Half-maximal effect was obtained at 1 nM. At mitogenic concentrations, VIP stimulated a marked accumulation (eightfold) of cAMP. In contrast to other growth-promoting neuropeptides, VIP did not induce an increase in cytosolic free Ca{sup 2+} or the activation of protein kinase C. The authors conclude that neuropeptides can modulate long-term cell proliferation through multiple signaling pathways.

Authors:
; ; ;  [1]
  1. (Imperial Cancer Research Fund, London (England))
Publication Date:
OSTI Identifier:
5321120
Resource Type:
Journal Article
Resource Relation:
Journal Name: Experimental Cell Research; (United States); Journal Volume: 175:2
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; AMP; RADIOIMMUNOASSAY; FIBROBLASTS; DNA REPLICATION; GROWTH FACTORS; RADIORECEPTOR ASSAY; PEPTIDES; BIOCHEMISTRY; BIOCHEMICAL REACTION KINETICS; CALCIUM COMPOUNDS; CATIONS; INSULIN; IODINE 125; MICE; PHOSPHOTRANSFERASES; SYNERGISM; THYMIDINE; TRITIUM COMPOUNDS; ALKALINE EARTH METAL COMPOUNDS; ANIMAL CELLS; ANIMALS; AZINES; BETA DECAY RADIOISOTOPES; BIOASSAY; CHARGED PARTICLES; CHEMISTRY; CONNECTIVE TISSUE CELLS; DAYS LIVING RADIOISOTOPES; DIAGNOSTIC TECHNIQUES; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; HETEROCYCLIC COMPOUNDS; HORMONES; HYDROGEN COMPOUNDS; IMMUNOASSAY; IMMUNOLOGY; INTERMEDIATE MASS NUCLEI; INTERNAL CONVERSION RADIOISOTOPES; IODINE ISOTOPES; IONS; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; MAMMALS; MITOGENS; NUCLEI; NUCLEIC ACID REPLICATION; NUCLEOSIDES; NUCLEOTIDES; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PEPTIDE HORMONES; PHOSPHORUS-GROUP TRANSFERASES; PROTEINS; PYRIMIDINES; RADIOASSAY; RADIOIMMUNODETECTION; RADIOIMMUNOLOGY; RADIOISOTOPES; REACTION KINETICS; RIBOSIDES; RODENTS; SOMATIC CELLS; TRACER TECHNIQUES; TRANSFERASES; VERTEBRATES; 550601* - Medicine- Unsealed Radionuclides in Diagnostics

Citation Formats

Zurier, B.B., Kozma, M., Sinnett-Smith, J., and Rozengurt, E. Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, Ca sup 2+ , and protein kinase C. United States: N. p., 1988. Web. doi:10.1016/0014-4827(88)90129-2.
Zurier, B.B., Kozma, M., Sinnett-Smith, J., & Rozengurt, E. Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, Ca sup 2+ , and protein kinase C. United States. doi:10.1016/0014-4827(88)90129-2.
Zurier, B.B., Kozma, M., Sinnett-Smith, J., and Rozengurt, E. 1988. "Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, Ca sup 2+ , and protein kinase C". United States. doi:10.1016/0014-4827(88)90129-2.
@article{osti_5321120,
title = {Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, Ca sup 2+ , and protein kinase C},
author = {Zurier, B.B. and Kozma, M. and Sinnett-Smith, J. and Rozengurt, E.},
abstractNote = {Vasoactive intestinal peptide synergistically stimulated initiation of DNA synthesis in Swiss 3T3 cells. The peptide stimulated ({sup 3}H)thymidine incorporation in the presence of insulin and either forskolin or an inhibitor of cAMP phosphodiesterase in a concentration-dependent manner. Half-maximal effect was obtained at 1 nM. At mitogenic concentrations, VIP stimulated a marked accumulation (eightfold) of cAMP. In contrast to other growth-promoting neuropeptides, VIP did not induce an increase in cytosolic free Ca{sup 2+} or the activation of protein kinase C. The authors conclude that neuropeptides can modulate long-term cell proliferation through multiple signaling pathways.},
doi = {10.1016/0014-4827(88)90129-2},
journal = {Experimental Cell Research; (United States)},
number = ,
volume = 175:2,
place = {United States},
year = 1988,
month = 5
}
  • The T/sub 84/ colonic epithelial cell line was used to examine protein phosphorylation during neurohumoral stimulation of ion transport. T/sub 84/ cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure ion transport stimulated by vasoactive intestinal peptide. Maximal stimulation of active secretion occurred after 8-10 min of stimulation. Protein phosphorylation events accompanying stimulated secretion were detected using two-dimensional gel electrophoresis to resolve phosphoproteins from monolayers previously labeled using /sup 32/P/sub i/. Within 8 min of exposure to vasoactive intestinal peptide, several phosphorylation events were detected, including a two- to fivefold increase in /sup 32/P incorporation intomore » four soluble proteins with apparent molecular weights of 17,000, 18,000, 23,000, and 37,000. The same phosphorylation response occurs in monolayers stimulated by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that cAMP mediates these intracellular events. This study indicates that changes in protein phosphorylation accompany the secretory action of vasocactive intestinal peptide and suggests that T/sub 84/ cells offer a useful model for studying the possibility that such phosphorylation events regulate enterocyte ion transport.« less
  • We studied the effect of vasoactive intestinal peptide (VIP) on the output of 35S-labeled macromolecules from ferret tracheal explants either placed in beakers or suspended in modified Ussing chambers. In Ussing chamber experiments, the radiolabel precursor, sodium (35S)sulfate, and all drugs were placed on the submucosal side of the tissue. Washings were collected at 30-min intervals from the luminal side and were dialyzed to remove unbound 35S, leaving radiolabeled macromolecules. Vasoactive intestinal peptide at 3 X 10(-7) M stimulated bound 35S output by a mean of + 252.6% (n . 14). The VIP response was dose-dependent with a near maximalmore » response and a half maximal response at approximately 10(-6) M and 10(-8), M, respectively. The VIP effect was not inhibited by a mixture of tetrodotoxin, atropine, I-propranolol, and phentolamine. Vasoactive intestinal peptide had no effect on the electrical properties of the of the tissues. We conclude that VIP stimulates output of sulfated-macromolecules from ferret tracheal submucosal glands without stimulating ion transport. Our studies also suggest that VIP acts on submucosal glands via specific VIP receptors. Vasoactive intestinal peptide has been shown to increase intracellular levels of cyclic AMP, and we suggest that this may be the mechanism for its effect on the output of macromolecules. This mechanism may be important in the neural regulation of submucosal gland secretion.« less
  • Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA{sup {minus}}). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA{sup +} or PKA{sup {minus}} cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA{sup {minus}} cells. These data indicate that the basal activity of PKAmore » may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA{sup +} and PKA{sup {minus}} cells was accompanied by desensitization of functional responses. Exposure of PKA{sup +} cells to 10{sup {minus}7} M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 {plus minus} 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA{sup +} or PKA{sup {minus}} cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA{sup {minus}} cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA{sup +} or PKA{sup {minus}} cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%.« less
  • The actions of somatostatin and of the phorbol ester 4{beta}-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and {sup 86}Rb{sup +} flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K{sup +} channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive {sup 86}Rb{sup +} efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, ofmore » protein kinase C, and of cAMP in the regulation of ATP-sensitive K{sup +} channels are discussed.« less
  • The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent withmore » increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA signaling pathway is involved downstream of cAMP. • Transcriptional MRP2 regulation ultimately involved participation of c-JUN and ATF2.« less