P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P-selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P-selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein-dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL-1 must interact with P-selectin in order for neutrophils to roll on P-selectin at physiological shear stresses.

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Letters to the Editor

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.4.c1437 ◽

1997 ◽

Vol 273 (4) ◽

pp. C1437-C1439

Author(s):

A. W. Cuthbert

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Single Channel ◽

Letters To The Editor ◽

High Affinity ◽

Channel Proteins ◽

Affinity Ligand ◽

Ligand Binding Sites ◽

Kinetics Of ◽

High Affinity Ligand

The following is the abstract of the article discussed in the subsequent letter: Blazer-Yost, Bonnie L., and Sandy I. Helman.The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities. Am. J. Physiol. 272 ( Cell Physiol. 41): C761–C769, 1997.—The amiloride-sensitive Na+ channel found in many transporting epithelia plays a key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and quantitate this important channel. Among biophysical methods, there is agreement as to the single-channel conductance and gating kinetics of the highly selective Na+ channel found in native epithelia. Amiloride and its analogs inhibit transport through the channel by binding to high-affinity ligand-binding sites. This characteristic of high-affinity binding has been used biochemically to quantitate channel densities and to isolate presumptive channel proteins. Although the goals of biophysical and biochemical experiments are the same in elucidating mechanisms underlying regulation of Na+transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and characterize the channel proteins.

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The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.3.c761 ◽

1997 ◽

Vol 272 (3) ◽

pp. C761-C769 ◽

Cited By ~ 26

Author(s):

B. L. Blazer-Yost ◽

S. I. Helman

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Single Channel ◽

Na Channel ◽

High Affinity ◽

Channel Proteins ◽

Affinity Ligand ◽

Kinetics Of ◽

High Affinity Ligand ◽

Epithelial Na Channel

The amiloride-sensitive Na+ channel found in many transporting epithelia plays a key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and quantitate this important channel. Among biophysical methods, there is agreement as to the single-channel conductance and gating kinetics of the highly selective Na+ channel found in native epithelia. Amiloride and its analogs inhibit transport through the channel by binding to high-affinity ligand-binding sites. This characteristic of high-affinity binding has been used biochemically to quantitate channel densities and to isolate presumptive channel proteins. Although the goals of biophysical and biochemical experiments are the same in elucidating mechanisms underlying regulation of Na+ transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and characterize the channel proteins.

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