[3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

K. N. Hawkins, R. J. Knapp, G. K. Lui, K. Gulya, W. Kazmierski, Y. P. Wan, J. T. Pelton, V. J. Hruby, I. Yamamura

Research output: Research - peer-reviewArticle

  • 99 Citations

Abstract

The cyclic, conformationally restricted octapeptide [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 108 M-1 and 2.49 x 108 M-1 min-1 at 25 and 37°C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10-2 min-1 and 1.03 x 10-1 min-1 at 25 and 37°C, respectively. Saturation isotherms of [3H]CTOP binding to rat brain membranes gave apparent K(d) values of 0.16 and 0.41 nM at 25 and 37°C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37°C, respectively. [3H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [3H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [3H]-[D-Pen2, D-Pen5]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [3H]CTOP binding was inhibited by a large number of opioid or opiate ligands. Putative mu opioid receptor ligands such as naltrexone, naloxone, CTOP and Tyr-D-Ala-Gly-N-MePhe-Gly-ol inhibited specific [3H]CTOP binding with high affinity. Other ligands, including delta and kappa opioids, somatostatin, substance P, alpha and beta adrenergic, dopaminergic as well as cholinergic ligands were much less potent or were ineffective. The density of [3H]CTOP binding sites in different regions of the central nervous system is consistent with reports of the regional distribution of mu receptors in the rat brain. These data show that [3H]CTOP is a potent and selective ligand and may be useful for further characterization of mu opioid receptors.

LanguageEnglish (US)
Pages73-80
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume248
Issue number1
StatePublished - 1989

Fingerprint

mu Opioid Receptor
Peptides
Brain
phenylalanyl-cyclo(cysteinyltyrosyl-tryptophyl-ornithyl-threonyl-penicillamine)threoninamide
Ligands
Membranes
delta Opioid Receptor
Naloxone
Opioid Analgesics
Binding Sites
Opiate Alkaloids
alanylglycine
Guanylyl Imidodiphosphate
Naltrexone
Enkephalins
Substance P
Somatostatin
Adrenergic Agents
Cholinergic Agents
Central Nervous System

ASJC Scopus subject areas

  • Pharmacology

Cite this

[3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain. / Hawkins, K. N.; Knapp, R. J.; Lui, G. K.; Gulya, K.; Kazmierski, W.; Wan, Y. P.; Pelton, J. T.; Hruby, V. J.; Yamamura, I.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 248, No. 1, 1989, p. 73-80.

Research output: Research - peer-reviewArticle

Hawkins, K. N. ; Knapp, R. J. ; Lui, G. K. ; Gulya, K. ; Kazmierski, W. ; Wan, Y. P. ; Pelton, J. T. ; Hruby, V. J. ; Yamamura, I./ [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain. In: Journal of Pharmacology and Experimental Therapeutics. 1989 ; Vol. 248, No. 1. pp. 73-80
@article{a2a8485ad56e47d6bdfad5f78e723185,
title = "[3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain",
abstract = "The cyclic, conformationally restricted octapeptide [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 108 M-1 and 2.49 x 108 M-1 min-1 at 25 and 37°C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10-2 min-1 and 1.03 x 10-1 min-1 at 25 and 37°C, respectively. Saturation isotherms of [3H]CTOP binding to rat brain membranes gave apparent K(d) values of 0.16 and 0.41 nM at 25 and 37°C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37°C, respectively. [3H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [3H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [3H]-[D-Pen2, D-Pen5]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [3H]CTOP binding was inhibited by a large number of opioid or opiate ligands. Putative mu opioid receptor ligands such as naltrexone, naloxone, CTOP and Tyr-D-Ala-Gly-N-MePhe-Gly-ol inhibited specific [3H]CTOP binding with high affinity. Other ligands, including delta and kappa opioids, somatostatin, substance P, alpha and beta adrenergic, dopaminergic as well as cholinergic ligands were much less potent or were ineffective. The density of [3H]CTOP binding sites in different regions of the central nervous system is consistent with reports of the regional distribution of mu receptors in the rat brain. These data show that [3H]CTOP is a potent and selective ligand and may be useful for further characterization of mu opioid receptors.",
author = "Hawkins, {K. N.} and Knapp, {R. J.} and Lui, {G. K.} and K. Gulya and W. Kazmierski and Wan, {Y. P.} and Pelton, {J. T.} and Hruby, {V. J.} and I. Yamamura",
year = "1989",
volume = "248",
pages = "73--80",
journal = "Journal of Pharmacology and Experimental Therapeutics",
issn = "0022-3565",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "1",

}

TY - JOUR

T1 - [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

AU - Hawkins,K. N.

AU - Knapp,R. J.

AU - Lui,G. K.

AU - Gulya,K.

AU - Kazmierski,W.

AU - Wan,Y. P.

AU - Pelton,J. T.

AU - Hruby,V. J.

AU - Yamamura,I.

PY - 1989

Y1 - 1989

N2 - The cyclic, conformationally restricted octapeptide [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 108 M-1 and 2.49 x 108 M-1 min-1 at 25 and 37°C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10-2 min-1 and 1.03 x 10-1 min-1 at 25 and 37°C, respectively. Saturation isotherms of [3H]CTOP binding to rat brain membranes gave apparent K(d) values of 0.16 and 0.41 nM at 25 and 37°C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37°C, respectively. [3H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [3H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [3H]-[D-Pen2, D-Pen5]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [3H]CTOP binding was inhibited by a large number of opioid or opiate ligands. Putative mu opioid receptor ligands such as naltrexone, naloxone, CTOP and Tyr-D-Ala-Gly-N-MePhe-Gly-ol inhibited specific [3H]CTOP binding with high affinity. Other ligands, including delta and kappa opioids, somatostatin, substance P, alpha and beta adrenergic, dopaminergic as well as cholinergic ligands were much less potent or were ineffective. The density of [3H]CTOP binding sites in different regions of the central nervous system is consistent with reports of the regional distribution of mu receptors in the rat brain. These data show that [3H]CTOP is a potent and selective ligand and may be useful for further characterization of mu opioid receptors.

AB - The cyclic, conformationally restricted octapeptide [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 108 M-1 and 2.49 x 108 M-1 min-1 at 25 and 37°C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10-2 min-1 and 1.03 x 10-1 min-1 at 25 and 37°C, respectively. Saturation isotherms of [3H]CTOP binding to rat brain membranes gave apparent K(d) values of 0.16 and 0.41 nM at 25 and 37°C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37°C, respectively. [3H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [3H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [3H]-[D-Pen2, D-Pen5]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [3H]CTOP binding was inhibited by a large number of opioid or opiate ligands. Putative mu opioid receptor ligands such as naltrexone, naloxone, CTOP and Tyr-D-Ala-Gly-N-MePhe-Gly-ol inhibited specific [3H]CTOP binding with high affinity. Other ligands, including delta and kappa opioids, somatostatin, substance P, alpha and beta adrenergic, dopaminergic as well as cholinergic ligands were much less potent or were ineffective. The density of [3H]CTOP binding sites in different regions of the central nervous system is consistent with reports of the regional distribution of mu receptors in the rat brain. These data show that [3H]CTOP is a potent and selective ligand and may be useful for further characterization of mu opioid receptors.

UR - http://www.scopus.com/inward/record.url?scp=0024477143&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024477143&partnerID=8YFLogxK

M3 - Article

VL - 248

SP - 73

EP - 80

JO - Journal of Pharmacology and Experimental Therapeutics

T2 - Journal of Pharmacology and Experimental Therapeutics

JF - Journal of Pharmacology and Experimental Therapeutics

SN - 0022-3565

IS - 1

ER -