Murine splenocytes express a naloxone-insensitive binding site for β-endorphin

Nahid A. Shahabi, Kristin M. Linner, Burt Sharp

Research output: Contribution to journalArticle

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Abstract

Naloxone-resistant binding sites for β-endor-phin have previously been observed on transformed peripheral blood mononuclear cells and on the EL4-thymoma cell line. These sites may be related to the naloxone-insensitive immunomodulatory effects of β-endorphin. The present study was performed 1) to determine whether these sites are present on normal splenocytes and 2) to characterize them. Ficoll-hypaque-purified murine splenocytes were used in a RRA with [125I] β-endorphin. Neither fresh intact cells obtained from viral antibody-free mice nor membrane preparations showed evidence of binding. How-ever, splenocytes cultured in 5% fetal bovine serum for 24-96 h showed sites on intact cells or membranes (after 3 h in culture no sites were present). Intact cultured splenocytes demonstrated a saturable binding isotherm, and Scatchard analysis showed a single site (Kd= 4.1 X 10-9 M). Competition studies showed that N-acetyl-β-endorphin (N-Ac-β-endorphin)-(1-31) was equipotent to ?-endorphin-(1-31). β-Endorphin-(6-31) and β-endor-phin-(28-31) were approximately 10- and 1000-fold less potent, respectively, whereas β-endorphin-(1-27) and naloxone were completely ineffective. Covalent cross-linking of [125I] β-endor-phin to splenocytes and resolution by gel electrophoresis showed bands at 66K and 57K which were displaced equipotently by increasing amounts of (8-endorphin and N-Ac-β-endorphin. β-Endorphin-(18-31) or (28-31) were less potent, and naloxone or other opioid ligands selective for receptor subtypes were ineffective. Thus, high affinity, naloxone-insensitive binding sites for (β-endorphin, which show competition characteristics distinctively different from brain opiate receptors, are inducible on normal mouse splenocytes. N-Ac-β-endorphin, presumed to be an inactivation product of β-endorphin because it fails to bind brain opiate receptors, may be functional at this naloxone-insensitive binding site.

Original languageEnglish (US)
Pages (from-to)1442-1448
Number of pages7
JournalEndocrinology
Volume126
Issue number3
DOIs
StatePublished - Jan 1 1990

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Endorphins
Naloxone
Binding Sites
Opioid Receptors
Viral Antibodies
Diatrizoate
Ficoll
Thymoma
Brain
Opioid Analgesics
Electrophoresis
Blood Cells

All Science Journal Classification (ASJC) codes

  • Endocrinology

Cite this

Murine splenocytes express a naloxone-insensitive binding site for β-endorphin. / Shahabi, Nahid A.; Linner, Kristin M.; Sharp, Burt.

In: Endocrinology, Vol. 126, No. 3, 01.01.1990, p. 1442-1448.

Research output: Contribution to journalArticle

Shahabi, Nahid A. ; Linner, Kristin M. ; Sharp, Burt. / Murine splenocytes express a naloxone-insensitive binding site for β-endorphin. In: Endocrinology. 1990 ; Vol. 126, No. 3. pp. 1442-1448.
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title = "Murine splenocytes express a naloxone-insensitive binding site for β-endorphin",
abstract = "Naloxone-resistant binding sites for β-endor-phin have previously been observed on transformed peripheral blood mononuclear cells and on the EL4-thymoma cell line. These sites may be related to the naloxone-insensitive immunomodulatory effects of β-endorphin. The present study was performed 1) to determine whether these sites are present on normal splenocytes and 2) to characterize them. Ficoll-hypaque-purified murine splenocytes were used in a RRA with [125I] β-endorphin. Neither fresh intact cells obtained from viral antibody-free mice nor membrane preparations showed evidence of binding. How-ever, splenocytes cultured in 5{\%} fetal bovine serum for 24-96 h showed sites on intact cells or membranes (after 3 h in culture no sites were present). Intact cultured splenocytes demonstrated a saturable binding isotherm, and Scatchard analysis showed a single site (Kd= 4.1 X 10-9 M). Competition studies showed that N-acetyl-β-endorphin (N-Ac-β-endorphin)-(1-31) was equipotent to ?-endorphin-(1-31). β-Endorphin-(6-31) and β-endor-phin-(28-31) were approximately 10- and 1000-fold less potent, respectively, whereas β-endorphin-(1-27) and naloxone were completely ineffective. Covalent cross-linking of [125I] β-endor-phin to splenocytes and resolution by gel electrophoresis showed bands at 66K and 57K which were displaced equipotently by increasing amounts of (8-endorphin and N-Ac-β-endorphin. β-Endorphin-(18-31) or (28-31) were less potent, and naloxone or other opioid ligands selective for receptor subtypes were ineffective. Thus, high affinity, naloxone-insensitive binding sites for (β-endorphin, which show competition characteristics distinctively different from brain opiate receptors, are inducible on normal mouse splenocytes. N-Ac-β-endorphin, presumed to be an inactivation product of β-endorphin because it fails to bind brain opiate receptors, may be functional at this naloxone-insensitive binding site.",
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N2 - Naloxone-resistant binding sites for β-endor-phin have previously been observed on transformed peripheral blood mononuclear cells and on the EL4-thymoma cell line. These sites may be related to the naloxone-insensitive immunomodulatory effects of β-endorphin. The present study was performed 1) to determine whether these sites are present on normal splenocytes and 2) to characterize them. Ficoll-hypaque-purified murine splenocytes were used in a RRA with [125I] β-endorphin. Neither fresh intact cells obtained from viral antibody-free mice nor membrane preparations showed evidence of binding. How-ever, splenocytes cultured in 5% fetal bovine serum for 24-96 h showed sites on intact cells or membranes (after 3 h in culture no sites were present). Intact cultured splenocytes demonstrated a saturable binding isotherm, and Scatchard analysis showed a single site (Kd= 4.1 X 10-9 M). Competition studies showed that N-acetyl-β-endorphin (N-Ac-β-endorphin)-(1-31) was equipotent to ?-endorphin-(1-31). β-Endorphin-(6-31) and β-endor-phin-(28-31) were approximately 10- and 1000-fold less potent, respectively, whereas β-endorphin-(1-27) and naloxone were completely ineffective. Covalent cross-linking of [125I] β-endor-phin to splenocytes and resolution by gel electrophoresis showed bands at 66K and 57K which were displaced equipotently by increasing amounts of (8-endorphin and N-Ac-β-endorphin. β-Endorphin-(18-31) or (28-31) were less potent, and naloxone or other opioid ligands selective for receptor subtypes were ineffective. Thus, high affinity, naloxone-insensitive binding sites for (β-endorphin, which show competition characteristics distinctively different from brain opiate receptors, are inducible on normal mouse splenocytes. N-Ac-β-endorphin, presumed to be an inactivation product of β-endorphin because it fails to bind brain opiate receptors, may be functional at this naloxone-insensitive binding site.

AB - Naloxone-resistant binding sites for β-endor-phin have previously been observed on transformed peripheral blood mononuclear cells and on the EL4-thymoma cell line. These sites may be related to the naloxone-insensitive immunomodulatory effects of β-endorphin. The present study was performed 1) to determine whether these sites are present on normal splenocytes and 2) to characterize them. Ficoll-hypaque-purified murine splenocytes were used in a RRA with [125I] β-endorphin. Neither fresh intact cells obtained from viral antibody-free mice nor membrane preparations showed evidence of binding. How-ever, splenocytes cultured in 5% fetal bovine serum for 24-96 h showed sites on intact cells or membranes (after 3 h in culture no sites were present). Intact cultured splenocytes demonstrated a saturable binding isotherm, and Scatchard analysis showed a single site (Kd= 4.1 X 10-9 M). Competition studies showed that N-acetyl-β-endorphin (N-Ac-β-endorphin)-(1-31) was equipotent to ?-endorphin-(1-31). β-Endorphin-(6-31) and β-endor-phin-(28-31) were approximately 10- and 1000-fold less potent, respectively, whereas β-endorphin-(1-27) and naloxone were completely ineffective. Covalent cross-linking of [125I] β-endor-phin to splenocytes and resolution by gel electrophoresis showed bands at 66K and 57K which were displaced equipotently by increasing amounts of (8-endorphin and N-Ac-β-endorphin. β-Endorphin-(18-31) or (28-31) were less potent, and naloxone or other opioid ligands selective for receptor subtypes were ineffective. Thus, high affinity, naloxone-insensitive binding sites for (β-endorphin, which show competition characteristics distinctively different from brain opiate receptors, are inducible on normal mouse splenocytes. N-Ac-β-endorphin, presumed to be an inactivation product of β-endorphin because it fails to bind brain opiate receptors, may be functional at this naloxone-insensitive binding site.

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