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Title: Butyrate modulates the expression of. beta. -adrenergic receptor subtype in 3T3-L1 cells

Abstract

In mouse 3T3-L1 fibroblasts, the glucocorticoid dexamethasone (dex) affects a switch in ..beta..-adrenergic receptor (..beta..AR) subtype expression from ..beta../sub 1/AR to ..beta../sub 2/AR and increases total ..beta..AR number. They now demonstrate a similar effect by sodium butyrate (B) and find that the combined effect of these two gene-activating agents is greater than additive suggesting different mechanisms of action on the ..beta..AR. ..beta..AR are assayed in membranes prepared from 3T3-L1 cells using the radiolabeled ..beta..AR-specific antagonist (/sup 125/I)-cyanopindolol. ..beta..AR subtype is determined by competition binding of the ..beta../sub 2/AR-selective antagonist ICI 118.551 for the radioligand. B (2-10mM) causes a dose-dependent increase in total ..beta..AR number (up to 2-fold over control) and the proportion of ..beta../sub 2/AR. B (5mM) causes a time-dependent increase in total ..beta..AR number (2-fold) and the proportion of ..beta../sub 2/AR up to 24 hr. Dex maximally increases total ..beta..AR number (2-fold) when treated for 48 hr at concentrations greater than or equal to 100nM. B (2 or 5mM) together with dex (250nM) have a greater than additive effect on total ..beta..AR number at 24 hr (1.7-fold) and at 48 hr (1.4-2.4-fold, using 5 or 10mM B and dex greater than or equal to 10nM). The proportion of ..beta../submore » 2/AR is also greater when both compounds are added together. In comparison with proprionate and valerate, B increases total ..beta..AR number and the proportion of ..beta../sub 2/AR to a greater extent and at lower concentrations. To determine a functional correlate to these findings, cells were pre-treated for 48 hr with B and/or dex, intracellular ATP labeled with /sup 3/H-adenine, followed by treatment with forskolin (10..mu..M) and ..beta..AR agonists. B caused a dramatic increase in /sup 3/H-cAMP produced compared to control and dex treatments and a greater than additive effect was again achieved when B and dex were added together.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Smith Kline and French Labs., Philadelphia, PA
OSTI Identifier:
6763914
Alternate Identifier(s):
OSTI ID: 6763914
Report Number(s):
CONF-8604222-
Journal ID: CODEN: FEPRA; TRN: 87-010336
Resource Type:
Conference
Resource Relation:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States); Journal Volume: 45:4; Conference: 70. annual meeting of the Federation of American Society for Experimental Biology, St. Louis, MO, USA, 13 Apr 1986
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BUTYRIC ACID; BIOCHEMICAL REACTION KINETICS; SYMPATHOMIMETICS; RECEPTORS; CELL MEMBRANES; DOSE-RESPONSE RELATIONSHIPS; FIBROBLASTS; IODINE 125; LIGANDS; MICE; TIME DEPENDENCE; TRACER TECHNIQUES; TRITIUM COMPOUNDS; ANIMAL CELLS; ANIMALS; AUTONOMIC NERVOUS SYSTEM AGENTS; BETA DECAY RADIOISOTOPES; CARBOXYLIC ACIDS; CELL CONSTITUENTS; CONNECTIVE TISSUE CELLS; DAYS LIVING RADIOISOTOPES; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; LABELLED COMPOUNDS; MAMMALS; MEMBRANE PROTEINS; MEMBRANES; MONOCARBOXYLIC ACIDS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; PROTEINS; RADIOISOTOPES; REACTION KINETICS; RODENTS; SOMATIC CELLS; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Poksay, K.S., Nakada, M.T., Crooke, S.T., and Stadel, J.M.. Butyrate modulates the expression of. beta. -adrenergic receptor subtype in 3T3-L1 cells. United States: N. p., 1986. Web.
Poksay, K.S., Nakada, M.T., Crooke, S.T., & Stadel, J.M.. Butyrate modulates the expression of. beta. -adrenergic receptor subtype in 3T3-L1 cells. United States.
Poksay, K.S., Nakada, M.T., Crooke, S.T., and Stadel, J.M.. Wed . "Butyrate modulates the expression of. beta. -adrenergic receptor subtype in 3T3-L1 cells". United States. doi:.
@article{osti_6763914,
title = {Butyrate modulates the expression of. beta. -adrenergic receptor subtype in 3T3-L1 cells},
author = {Poksay, K.S. and Nakada, M.T. and Crooke, S.T. and Stadel, J.M.},
abstractNote = {In mouse 3T3-L1 fibroblasts, the glucocorticoid dexamethasone (dex) affects a switch in ..beta..-adrenergic receptor (..beta..AR) subtype expression from ..beta../sub 1/AR to ..beta../sub 2/AR and increases total ..beta..AR number. They now demonstrate a similar effect by sodium butyrate (B) and find that the combined effect of these two gene-activating agents is greater than additive suggesting different mechanisms of action on the ..beta..AR. ..beta..AR are assayed in membranes prepared from 3T3-L1 cells using the radiolabeled ..beta..AR-specific antagonist (/sup 125/I)-cyanopindolol. ..beta..AR subtype is determined by competition binding of the ..beta../sub 2/AR-selective antagonist ICI 118.551 for the radioligand. B (2-10mM) causes a dose-dependent increase in total ..beta..AR number (up to 2-fold over control) and the proportion of ..beta../sub 2/AR. B (5mM) causes a time-dependent increase in total ..beta..AR number (2-fold) and the proportion of ..beta../sub 2/AR up to 24 hr. Dex maximally increases total ..beta..AR number (2-fold) when treated for 48 hr at concentrations greater than or equal to 100nM. B (2 or 5mM) together with dex (250nM) have a greater than additive effect on total ..beta..AR number at 24 hr (1.7-fold) and at 48 hr (1.4-2.4-fold, using 5 or 10mM B and dex greater than or equal to 10nM). The proportion of ..beta../sub 2/AR is also greater when both compounds are added together. In comparison with proprionate and valerate, B increases total ..beta..AR number and the proportion of ..beta../sub 2/AR to a greater extent and at lower concentrations. To determine a functional correlate to these findings, cells were pre-treated for 48 hr with B and/or dex, intracellular ATP labeled with /sup 3/H-adenine, followed by treatment with forskolin (10..mu..M) and ..beta..AR agonists. B caused a dramatic increase in /sup 3/H-cAMP produced compared to control and dex treatments and a greater than additive effect was again achieved when B and dex were added together.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:4,
place = {United States},
year = {Wed Mar 05 00:00:00 EST 1986},
month = {Wed Mar 05 00:00:00 EST 1986}
}

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  • Mouse 3T3-L1 fibroblasts contain beta-adrenergic receptors (BAR), predominantly of the B/sub 1/ subtype. Incubation of these cells with 2-10 mM sodium butyrate (SB) for 24-48 hr results in a switch in the BAR subtype from B/sub 1/ to B/sub 2/ and promotes a 1.5 to 2.5 fold increase in total BAR number. Other short chain acids were not as effective as SB in promoting changes in BAR. BAR were assayed in membranes prepared from the 3T3-L1 cells using the radiolabeled antagonist (/sup 125/I)-cyanopindolol and the B/sub 2/ selective antagonist ICI 118.551. BAR subtype switch was confirmed functionally by measuring cellularmore » cAMP accumulation in response to agonists. The structure and amount of the alpha subunits of the guanine nucleotide regulatory proteins N/sub s/ and N/sub i/ were determined by ADP-ribosylation using /sup 32/P-NAD and either cholera toxin or pertussis toxin for labeling of the respective subunits. Preincubation of cells with 5 mM SB for 48 hr resulted in a 2-3 fold increase in the labeling of the alpha subunits of both N/sub s/ and N/sub i/. A protein of M/sub r/ = 44,000 showed enhanced labeling by cholera toxin following SB treatment of the cells. These data indicate SB concomitantly regulates expression of BAR subtype and components of the adenylate cyclase in 3T3-L1 cells.« less
  • 3T3-L1 fibroblasts, containing ..beta..-adrenergic receptors (..beta..AR) of predominantly the ..beta../sub 1/AR subtype, express only ..beta../sub 2/AR after 48 hr treatment with 250 nM dexamethasone (dex). A two-fold increase in ..beta..AR number was also observed upon dex treatment. ..beta..AR were assayed in membranes using the radioligand (/sup 125/I)-cyanopindolol and the ..beta../sub 2/AR selective antagonist ICI. The relative potency of agonists in stimulating whole cell adenylate cyclase activity was consistent with the subtypes determined by binding experiments. The ability of compounds to cause the subtype switch correlates with glucocorticoid activity since these compounds were more effective in facilitating the subtype conversion andmore » the increase in ..beta..AR number than mineralocorticoids. Sex steroids and thyroid hormone were ineffective at concentrations up to 1..mu..M in causing these changes. RNA and protein synthesis appear to be required for the ..beta..AR subtype switch and increase in ..beta..AR number since these changes were not observed with dex treatment in the presence of 1..mu..g/ml actinomycin D or 1..mu..g/ml cyclohexamide. This study suggests that glucocorticoids may induce gene activation in 3T3-L1 fibroblasts that results in an increase in ..beta..AR number and a shift in subtype.« less
  • The gene encoding the human beta-adrenergic receptor (BAR) was expressed in mammalian cells and the binding properties of the expressed protein examined. The human BAR could be expressed at high levels in mammalian cells and was able to mediate the stimulation of adenylate cyclase when expressed in a stable cell line. The expressed receptor was found to bind the radiolabeled antagonist /sup 125/I-cyano-pindolol with a high affinity, comparable to that observed for native receptor in a variety of systems and to the hamster cloned BAR expressed in the same cells. The pharmacology of the receptor was investigated in membranes preparedmore » from the cells. Agonist competition binding showed the human BAR to be of the beta-2 subtype, with isoproterenol > epinephrine > norepinephrine in binding to the receptor. EC50 values for the three agonists were 0.2 ..mu..M, 1 ..mu..M, and 10 ..mu..M, respectively. This is in agreement with their findings for the cloned BAR from hamster, which shares sequence homology with the human protein.« less
  • Cultured 3T3-L1 cells have been used as a model system to investigate the mechanism of fatty acid uptake by adipose tissue. Using a 1:1 molar ratio of /sup 14/C-oleate and defatted bovine serum albumin (BSA), fatty acid (FA) uptake was quantitated at 4/sup 0/ and 37/sup 0/ as cell associated radioactivity. The profile of FA uptake in preadipocytes and adipocytes was biphasic; an initial rapid phase (1-20s) followed by a second slower phase (60-480s). At 37/sup 0/ the initial rate of FA accumulation in preadipocytes was identical to that in adipocytes, whereas the rate of accumulation during the second phasemore » increased 7-fold (100 ..mu..M total FA) as a consequence of adipose conversion. When uptake measurements were made at 4/sup 0/ in adipocytes, the initial rate was identical to that at 37/sup 0/, however the rate of second phase decreased 5-fold. Incubation of /sup 14/C-BSA and nonradiolabeled FA with adipocyte monolayers (100 ..mu..M total FA) resulted in the rapid association (t/sub 1/2/ = 20s) of the BSA-FA complex with the cell surface. Incubation of 100, 10, and 1 ..mu..M total FA with adipocytes resulted in a 50-fold change in FA accumulation during the second phase. These results suggest that (1) FA uptake is significantly increased after differentiation, suggesting the participation of specialized proteins, (2) the temperature-insensitive initial FA accumulation can be attributed to rapid association of the BSA-FA complex to the cell surface, (3) the second phase of FA accumulation represents uptake.« less
  • An /alpha//sub 2/-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet /alpha//sub 2/-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet /alpha//sub 2/-adrenergic receptor and is consistent with the structure of other members of he family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the /alpha//sub 2/-adrenergic ligand (/sup 3/H)rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the /alpha//sub 2/B-adrenergic receptor. The gene for this receptor ismore » on human chromosome 4, whereas the gene for the human platelet /alpha//sub 2/-adrenergic receptor (/alpha//sub 2/A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective /alpha/-adrenergic ligands.« less