[3H]leukotriene B4 binding to the guinea-pig spleen membrane preparation: A rich tissue source for a high-affinity leukotriene B4 receptor site

J. B. Cheng, E. I P Cheng, F. Kohi, R. G. Townley

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Abstract

Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [3H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compte with [3H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [3H]LTB4 binding, but NaCl and KCl decreased it. Spleen [3H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25°C was 0.47 nM-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg protein. Moreover, the LTB4/[3H]LTB4 competition study performed at 4 or 25° C revealed the inhibitory constant (K(i)) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [3H]LTB4 binding was: LTB4 (K(i) = 2.8 nM) > 20-hydroxyl-LTB4 (23 nM) > LTA4 (48 nM) > LTA4 methyl ester (0.13 μM) > 20-carboxyl-LTB4 (>6.6 μM) ≥ arachidonic acid (0.15 mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihydropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [3H]LTB4 binding. Comparison of the ability of LTB4 to inhibit [3H]LTB4 binding among various tissue homogenates demonstrated the following tissue rank for its effect: spleen > thymus gland > lung ≥ uterus = urinary bladder = brain = adrenal gland = small intestine = liver = kidney = heart. The low-affinity binding of [3H]LTB4 to the guinea-pig granulcoyte argues against the the concept that binding to this cell accounts for the [3H]LTB4 high-affinity binding site in the spleen. We conclude that guinea-pig spleen contains a rich tissue source of [3H]LTB4 receptor high-affinity binding sites and suggest that these binding sites may play a role in immunoregulation.

Original languageEnglish
Pages (from-to)126-132
Number of pages7
JournalJournal of Pharmacology and Experimental Therapeutics
Volume236
Issue number1
StatePublished - 1986

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Leukotriene B4 Receptors
Leukotriene B4
Guinea Pigs
Spleen
Membranes
Binding Sites
Leukotriene A4
Felodipine

All Science Journal Classification (ASJC) codes

  • Pharmacology

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[3H]leukotriene B4 binding to the guinea-pig spleen membrane preparation : A rich tissue source for a high-affinity leukotriene B4 receptor site. / Cheng, J. B.; Cheng, E. I P; Kohi, F.; Townley, R. G.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 236, No. 1, 1986, p. 126-132.

Research output: Contribution to journalArticle

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title = "[3H]leukotriene B4 binding to the guinea-pig spleen membrane preparation: A rich tissue source for a high-affinity leukotriene B4 receptor site",
abstract = "Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [3H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compte with [3H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [3H]LTB4 binding, but NaCl and KCl decreased it. Spleen [3H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25°C was 0.47 nM-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg protein. Moreover, the LTB4/[3H]LTB4 competition study performed at 4 or 25° C revealed the inhibitory constant (K(i)) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [3H]LTB4 binding was: LTB4 (K(i) = 2.8 nM) > 20-hydroxyl-LTB4 (23 nM) > LTA4 (48 nM) > LTA4 methyl ester (0.13 μM) > 20-carboxyl-LTB4 (>6.6 μM) ≥ arachidonic acid (0.15 mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihydropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [3H]LTB4 binding. Comparison of the ability of LTB4 to inhibit [3H]LTB4 binding among various tissue homogenates demonstrated the following tissue rank for its effect: spleen > thymus gland > lung ≥ uterus = urinary bladder = brain = adrenal gland = small intestine = liver = kidney = heart. The low-affinity binding of [3H]LTB4 to the guinea-pig granulcoyte argues against the the concept that binding to this cell accounts for the [3H]LTB4 high-affinity binding site in the spleen. We conclude that guinea-pig spleen contains a rich tissue source of [3H]LTB4 receptor high-affinity binding sites and suggest that these binding sites may play a role in immunoregulation.",
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T2 - A rich tissue source for a high-affinity leukotriene B4 receptor site

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AU - Cheng, E. I P

AU - Kohi, F.

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N2 - Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [3H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compte with [3H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [3H]LTB4 binding, but NaCl and KCl decreased it. Spleen [3H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25°C was 0.47 nM-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg protein. Moreover, the LTB4/[3H]LTB4 competition study performed at 4 or 25° C revealed the inhibitory constant (K(i)) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [3H]LTB4 binding was: LTB4 (K(i) = 2.8 nM) > 20-hydroxyl-LTB4 (23 nM) > LTA4 (48 nM) > LTA4 methyl ester (0.13 μM) > 20-carboxyl-LTB4 (>6.6 μM) ≥ arachidonic acid (0.15 mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihydropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [3H]LTB4 binding. Comparison of the ability of LTB4 to inhibit [3H]LTB4 binding among various tissue homogenates demonstrated the following tissue rank for its effect: spleen > thymus gland > lung ≥ uterus = urinary bladder = brain = adrenal gland = small intestine = liver = kidney = heart. The low-affinity binding of [3H]LTB4 to the guinea-pig granulcoyte argues against the the concept that binding to this cell accounts for the [3H]LTB4 high-affinity binding site in the spleen. We conclude that guinea-pig spleen contains a rich tissue source of [3H]LTB4 receptor high-affinity binding sites and suggest that these binding sites may play a role in immunoregulation.

AB - Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [3H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compte with [3H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [3H]LTB4 binding, but NaCl and KCl decreased it. Spleen [3H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25°C was 0.47 nM-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg protein. Moreover, the LTB4/[3H]LTB4 competition study performed at 4 or 25° C revealed the inhibitory constant (K(i)) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [3H]LTB4 binding was: LTB4 (K(i) = 2.8 nM) > 20-hydroxyl-LTB4 (23 nM) > LTA4 (48 nM) > LTA4 methyl ester (0.13 μM) > 20-carboxyl-LTB4 (>6.6 μM) ≥ arachidonic acid (0.15 mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihydropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [3H]LTB4 binding. Comparison of the ability of LTB4 to inhibit [3H]LTB4 binding among various tissue homogenates demonstrated the following tissue rank for its effect: spleen > thymus gland > lung ≥ uterus = urinary bladder = brain = adrenal gland = small intestine = liver = kidney = heart. The low-affinity binding of [3H]LTB4 to the guinea-pig granulcoyte argues against the the concept that binding to this cell accounts for the [3H]LTB4 high-affinity binding site in the spleen. We conclude that guinea-pig spleen contains a rich tissue source of [3H]LTB4 receptor high-affinity binding sites and suggest that these binding sites may play a role in immunoregulation.

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