Endothelium derived relaxing factor release from canine coronary artery by leukocytes.

Lectins, known to recognize endothelial cell adhesion molecules, have been shown to release endothelium-derived relaxing factor (EDRF) from blood vessels. We investigated the effects of different leukocyte-type cells to determine if these cells, by interacting with the endothelium, could release EDRF from the circumflex branch of the canine coronary artery. The following cells were investigated: human promyelocytic leukemia (HL-60), human monocyte (THP-1), and human Burkitt lymphoma (DAUDI). All of these cells produced a significant endothelium-dependent relaxation of the dog coronary artery in the presence of ibuprofen. The endothelium-dependent relaxations were reversed by hemoglobin (10 microM), methylene blue (3 microM), 6-anilino-5,8-quinolinedione (LY 83583, 30 microM), and NG-nitro-L-arginine methyl ester (L-NAME, 1 mM). HL-60 cells grown in the presence of 1 mM L-NAME retained their ability to cause endothelium-dependent relaxation of the canine coronary artery, suggesting that the source of the NO was the endothelium and not the HL-60 cells. The cell-induced vascular relaxation could be obtained in the absence of extracellular calcium. It is suggested that HL-60, THP-1, and DAUDI cells interact with a specific receptor on the endothelial cell and as a result of this interaction the endothelial cells are stimulated to release EDRF.

EDRF release from canine coronary artery by lectins.

1. The effect of wheat germ agglutinin (WGA), concanavalin A (Con A) and lentil lectin agglutinin (LCA) were investigated on pre-contracted canine coronary artery rings in vitro. 2. In endothelium-intact canine coronary artery, contracted with the thromboxane A2-analogue, U46619, WGA relaxed the tissue in a concentration-dependent manner, with an inhibitory concentration (IC50) of 112 +/- 17 nM (n = 6). In the absence of an endothelium, WGA did not cause any relaxation of the tissue. 3. In endothelium-intact canine coronary artery, contracted with the thromboxane A2-analogue, U46619. LCA relaxed the tissue in a concentration-dependent manner, with an inhibitory concentration (IC50) of 423.1 +/- 41 nM (n = 6). In the absence of an endothelium, LCA produced a 20.1 +/- 1.1% (n = 6) relaxation at the highest concentration tested (3 microM). 4. Concanavalin A (Con A) relaxed canine coronary artery in a partial endothelium-dependent manner with an IC50 of 104 +/- 19 nM on endothelium-intact coronary artery and an IC50 of 1.3 +/- 0.3 microM (n = 6) on endothelium-denuded tissues. 5. The relaxation effects of WGA were attenuated by 1 mM NG-monomethyl L-arginine (L-NMMA) and completely inhibited by haemoglobin (3 microM), methylene blue (10 microM) and LY 83583 (30 microM). Ibuprofen had no effect on WGA-induced relaxation. 6. The relaxant effects of WGA were reversed by addition of 20 mM N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-galactosamine (Ga1NAc) but not by alpha-mannose, D-(+)-galactose, and beta-lactose, whereas the endothelin-dependent relaxations to LCA and Con A were unaffected. 7.The endothelium-dependent relaxation induced by the lectins was unaffected by pretreatment of the tissue with 1 microM atropine.8. In the absence of extracellular calcium, WGA was also able to release EDRF suggesting that WGA acts through a second messenger system to release intracellular calcium.9. We suggest that WGA acts as an agonist to release EDRF from endothelial cells possibly by binding to a sugar moiety, specific receptor or adhesion molecules on the endothelial cell surface.

The effects of lectins on the release of EDRF from rabbit aorta.

The effects of the lectins, wheat germ agglutinin (WGA) and concanavalin A (Con A) on endothelium intact pre-contracted rabbit aorta were investigated. WGA (8.3 microM) produced endothelium-dependent relaxations, while Con A (4.3 microM) produced a partially endothelium-dependent relaxation. The endothelium-dependent relaxations to WGA were completely reversed by N-acetylglucosamine, haemoglobin and methylene blue and were partially reversed by NG-monomethyl-L-arginine (L-NMMA). It is suggested that WGA works as an agonist in releasing endothelium-derived relaxing factor (EDRF) from the endothelium of rabbit aorta by interacting with a glycosylated receptor on endothelial cells.

Evidence of glycosylated sites on the endothelin-1 receptor in Swiss 3T3 cells.

The effects of incubation of intact cells with six different lectins on the specific binding of [125I]endothelin-1 (ET-1) were determined in Swiss 3T3 fibroblasts. ET-1 binding was unaffected by pretreatment of cells for 1 h at 37 degrees C with concanavalin A, soybean agglutinin, Ulex europaeus agglutinin I, peanut agglutinin, or Galanthus nivalis agglutinin. However, preincubation of cells with 300 micrograms/ml of wheat germ agglutinin resulted in a 70% decrease in specific binding of ET-1 to cell-surface receptors. The inhibitory effects of wheat germ agglutinin were diminished by brief incubation of lectin-treated cells with 100 mM N-acetylglucosamine, a monosaccharide specifically recognized by wheat germ agglutinin. Neither glucose nor mannose had any effect on wheat germ agglutinin-mediated inhibition of the specific binding of ET-1. These results suggest that the ET-1 receptor on 3T3 cells is a glycoprotein that contains one or more N-acetylglucosamine residues at or near the ligand binding site.

Mobilization of intracellular calcium and stimulation of calcium fluxes by endothelin-2 in cultured mouse swiss 3T3 fibroblasts.

Specific receptor-induced signal transduction mechanisms for the endothelin-2 isoform (ET-2), a potent vasoconstrictor of vascular smooth muscle, were examined in Swiss 3T3 cells. Half-maximal binding (EC(50)) and maximal, saturable binding (B(max)) were estimated from Scatchard analyses and were found to be 24.2 +/- 3.3 pM and 56500 +/- 1700 sites/cells, respectively. A saturating concentration of ET-2 (100 nM) increased intracellular free calcium (measured by Fura-2 fluorescence) from a resting level of ? 100 nM to a peak level of 600-800 nM. The initial increase in intracellular free calcium was transitory and was followed by a smaller maintained elevation (?250 nM). In the absence of extracellular calcium, ET-2 induced a transitory response equal in size to the peak in the presence of extracellular calcium, but the maintained response was absent. ET-2 increased intracellular free calcium in a concentration-dependent manner with an EC(50) of ? 1 nM. In calcium free solution (2 mM EGTA), ET-2 increased the efflux of (45)Ca from cells loaded to isotopic equilibrium (3 h) with (45)Ca. The intracellular second messenger, IP(3), also increased the calcium efflux from saponin permeabilized 3T3 cells loaded with (45)Ca (pCa 6) in the presence of MgATP. In the presence of extracellular calcium, ET-2 significantly increased calcium uptake into 3T3 cells by 92 +/- 36.6 pmoles/million cells/2 min (n = 8). It is suggested that ET-2 binds to specific, high affinity receptors in 3T3 cells and that this receptor interaction increases the intracellular free calcium by IP(3)-induced mobilization of calcium from cellular stores and by increasing influx of extracellular calcium.

Receptor kinetics differ for endothelin-1 and endothelin-2 binding to Swiss 3T3 fibroblasts.

The equilibrium binding, kinetics of ligand-receptor interactions, and biological activity of endothelin-1 and -2 have been studied in Swiss 3T3 fibroblasts. Scatchard analyses of saturation binding data for ET-1 and -2, performed at 4 degrees C to prevent internalization of the occupied receptor, revealed similar affinity constants and numbers of binding sites for endothelin-1 and -2. Experiments designed to determine ligand-induced effects on 45Ca efflux demonstrated no qualitative or quantitative differences between the two endothelin isoforms. In contrast, kinetic studies resulted in different rates of dissociation for the two isoforms and different extents of dissociation. Specifically, only 40% of the bound [125I]endothelin-1 was dissociated at 4 h following the addition of excess unlabeled ligand, whereas 85-90% of the bound [125I]endothelin-2 was dissociated under the same conditions. Endothelin-1 and -2 also differed in the percent of specific cell-associated ligand bound after a 2 h incubation at 37 degrees C following an initial equilibration at 4 degrees C. The differences in dissociation rates and association or internalization rates at 37 degrees C are the first data that differentiate between the two isoforms. It is suggested that isoform-specific differences in the rate of dissociation from cell surface endothelin receptors influence the level of cell-associated endothelin and may be important in determining physiologic responses in vivo.

Turnover of functional basic fibroblast growth factor receptors on the surface of BHK and NIH 3T3 cells.

The recovery of functional cell-surface bFGF receptors after trypsin treatment was studied in BHK cells and NIH 3T3 cells. Restoration of functional bFGF receptors occurred at an approximately linear rate with 50% of the high-affinity binding capacity restored after 4 hr. Restoration of functional receptors required protein synthesis but not RNA synthesis. Upon exposure of BHK cells to bFGF, cell-surface receptors were rapidly lost, with only 25% remaining after 1 hr. When the bFGF was removed, down-regulated BHK and NIH 3T3 cells recovered cell-surface receptors at about the same rate observed in trypsin-treated cells. The recovery of receptors after down-regulation was inhibited by protein synthesis inhibitors. Addition of the protein synthesis inhibitor cycloheximide to unperturbed cultures of BHK or NIH 3T3 cells resulted in a time-dependent loss of cell-surface bFGF receptors, demonstrating that the receptors turn over constantly in the absence of ligand. These results suggest that bFGF receptors do not recycle and must be continuously synthesized.