Activation of protein kinase C rapidly down-regulates naloxone-resistant receptors for β-endorphin on U937 cells

N. A. Shahabi, Burt Sharp

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Abstract

Activation of protein kinase-C (PKC) has been reported to modify a variety of receptor-ligand interactions, including that of tumor necrosis factor α with immune cells. Thus, we studied the effect of phorbol esters on the binding of β-endorphin to naloxone-resistant receptors on the promonocyte-like U937 cell line. After incubating intact U937 cells with phorbol 12- myristate 13-acetate (PMA, 100 nM) at 22°C for 30 min, the specific binding of 125I-β-endorphin was maximally reduced by approximately 40%. Only PMA (10-150 nM), and not the biologically inactive phorbol, 4α-phorbol 12,13-didecanoate, caused this rapid, dose-dependent down-regulation. PMA did not interfere with the radioreceptor assay nor did it induce down-regulation when incubated with cell membrane. Scatchard analysis revealed that PMA significantly reduced both the number of receptors and K(d) (10,640 receptors/cell and K(d) = 2.9 ± 0.1 nM for control vs. 4,868 receptors/cell and K(d) = 1.5 ± 0.7 nM for 150 nM PMA). The effect of PMA was abolished by preincubating cells with the inhibitors of PKC, N-(2-aminoethyl)-5 isoquinolinesulfonamide or 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine. Down-regulation was reversible; removing 100 nM PMA from the media partially restored binding by 3 h and completely by 24 h. At 22°C, internalization of 125I-β-endorphin was not observed, and this was not altered by PMA. These results show that activation of PKC rapidly down-regulated the naloxone-resistant receptor for β-endorphin on intact U937 cells; the number of receptors available for interaction with the ligand was reduced by a mechanism independent of internalization.

Original languageEnglish (US)
Pages (from-to)276-281
Number of pages6
JournalJournal of Pharmacology and Experimental Therapeutics
Volume264
Issue number1
StatePublished - 1993

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Endorphins
U937 Cells
Protein Kinase C
Down-Regulation
Monocyte-Macrophage Precursor Cells
Ligands
Radioligand Assay
Phorbol Esters
Acetates
Tumor Necrosis Factor-alpha
Cell Membrane
Cell Line
naloxone receptor

All Science Journal Classification (ASJC) codes

  • Pharmacology

Cite this

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title = "Activation of protein kinase C rapidly down-regulates naloxone-resistant receptors for β-endorphin on U937 cells",
abstract = "Activation of protein kinase-C (PKC) has been reported to modify a variety of receptor-ligand interactions, including that of tumor necrosis factor α with immune cells. Thus, we studied the effect of phorbol esters on the binding of β-endorphin to naloxone-resistant receptors on the promonocyte-like U937 cell line. After incubating intact U937 cells with phorbol 12- myristate 13-acetate (PMA, 100 nM) at 22°C for 30 min, the specific binding of 125I-β-endorphin was maximally reduced by approximately 40{\%}. Only PMA (10-150 nM), and not the biologically inactive phorbol, 4α-phorbol 12,13-didecanoate, caused this rapid, dose-dependent down-regulation. PMA did not interfere with the radioreceptor assay nor did it induce down-regulation when incubated with cell membrane. Scatchard analysis revealed that PMA significantly reduced both the number of receptors and K(d) (10,640 receptors/cell and K(d) = 2.9 ± 0.1 nM for control vs. 4,868 receptors/cell and K(d) = 1.5 ± 0.7 nM for 150 nM PMA). The effect of PMA was abolished by preincubating cells with the inhibitors of PKC, N-(2-aminoethyl)-5 isoquinolinesulfonamide or 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine. Down-regulation was reversible; removing 100 nM PMA from the media partially restored binding by 3 h and completely by 24 h. At 22°C, internalization of 125I-β-endorphin was not observed, and this was not altered by PMA. These results show that activation of PKC rapidly down-regulated the naloxone-resistant receptor for β-endorphin on intact U937 cells; the number of receptors available for interaction with the ligand was reduced by a mechanism independent of internalization.",
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T1 - Activation of protein kinase C rapidly down-regulates naloxone-resistant receptors for β-endorphin on U937 cells

AU - Shahabi, N. A.

AU - Sharp, Burt

PY - 1993

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N2 - Activation of protein kinase-C (PKC) has been reported to modify a variety of receptor-ligand interactions, including that of tumor necrosis factor α with immune cells. Thus, we studied the effect of phorbol esters on the binding of β-endorphin to naloxone-resistant receptors on the promonocyte-like U937 cell line. After incubating intact U937 cells with phorbol 12- myristate 13-acetate (PMA, 100 nM) at 22°C for 30 min, the specific binding of 125I-β-endorphin was maximally reduced by approximately 40%. Only PMA (10-150 nM), and not the biologically inactive phorbol, 4α-phorbol 12,13-didecanoate, caused this rapid, dose-dependent down-regulation. PMA did not interfere with the radioreceptor assay nor did it induce down-regulation when incubated with cell membrane. Scatchard analysis revealed that PMA significantly reduced both the number of receptors and K(d) (10,640 receptors/cell and K(d) = 2.9 ± 0.1 nM for control vs. 4,868 receptors/cell and K(d) = 1.5 ± 0.7 nM for 150 nM PMA). The effect of PMA was abolished by preincubating cells with the inhibitors of PKC, N-(2-aminoethyl)-5 isoquinolinesulfonamide or 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine. Down-regulation was reversible; removing 100 nM PMA from the media partially restored binding by 3 h and completely by 24 h. At 22°C, internalization of 125I-β-endorphin was not observed, and this was not altered by PMA. These results show that activation of PKC rapidly down-regulated the naloxone-resistant receptor for β-endorphin on intact U937 cells; the number of receptors available for interaction with the ligand was reduced by a mechanism independent of internalization.

AB - Activation of protein kinase-C (PKC) has been reported to modify a variety of receptor-ligand interactions, including that of tumor necrosis factor α with immune cells. Thus, we studied the effect of phorbol esters on the binding of β-endorphin to naloxone-resistant receptors on the promonocyte-like U937 cell line. After incubating intact U937 cells with phorbol 12- myristate 13-acetate (PMA, 100 nM) at 22°C for 30 min, the specific binding of 125I-β-endorphin was maximally reduced by approximately 40%. Only PMA (10-150 nM), and not the biologically inactive phorbol, 4α-phorbol 12,13-didecanoate, caused this rapid, dose-dependent down-regulation. PMA did not interfere with the radioreceptor assay nor did it induce down-regulation when incubated with cell membrane. Scatchard analysis revealed that PMA significantly reduced both the number of receptors and K(d) (10,640 receptors/cell and K(d) = 2.9 ± 0.1 nM for control vs. 4,868 receptors/cell and K(d) = 1.5 ± 0.7 nM for 150 nM PMA). The effect of PMA was abolished by preincubating cells with the inhibitors of PKC, N-(2-aminoethyl)-5 isoquinolinesulfonamide or 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine. Down-regulation was reversible; removing 100 nM PMA from the media partially restored binding by 3 h and completely by 24 h. At 22°C, internalization of 125I-β-endorphin was not observed, and this was not altered by PMA. These results show that activation of PKC rapidly down-regulated the naloxone-resistant receptor for β-endorphin on intact U937 cells; the number of receptors available for interaction with the ligand was reduced by a mechanism independent of internalization.

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