Histamine degradative uptake by cultured human pulmonary vascular endothelial cells utilizes an inflammatory cell diamine oxidase.

Neutrophil-endothelial interactions, altered clearance properties of the lung toward vasoactive mediators, and damaging effects of histamine that target the lung represent prominent elements of the inflammatory response at the systemic level. The pulmonary vasculature is unusual in that, unlike other tissues and vascular beds, it normally does not metabolize circulating histamine in vivo, although histamine-metabolizing enzyme activities have been detected in disrupted lung tissue. We have therefore explored the capability of human pulmonary artery endothelial cells in culture to express the receptor-mediated histamine degradative uptake system we previously defined in systemic endothelial cells (Haddock, R. C., Mack, P., Leal, S., and Baenziger, N. L. (1990) J. Biol. Chem. 265, 14395-14401). Pulmonary endothelial cells display all components of this system: histamine methyltransferase generating the proximal cell-associated metabolite tele-methylhistamine and receptors binding diamine oxidase which generates the distal product methylimidazoleacetic acid that is accumulated by the cells. A diamine oxidase released from human neutrophil granules by activation with Ca2+ ionophore binds pulmonary and systemic endothelial cell and fibroblast diamine oxidase receptors and, thereby, participates in histamine degradative uptake. This enzyme utilizes cell-associated tele-methylhistamine as a substrate, preferentially generating methylimidazoleacetic acid in addition to reactive oxygen species. Thus the enzymatic and interactive cellular machinery for histamine clearance is inherently present as a functional unit in two major human pulmonary cell types. It interacts with products of inflammatory host defense cells, and pulmonary endothelial-neutrophil interactions via this pathway may influence the progression of inflammation.

An environmentally regulated receptor for diamine oxidase modulates human endothelial cell/fibroblast histamine degradative uptake.

Human vascular endothelial cells and fibroblasts express a cell-surface degradative pathway for the multifunctional mediator histamine, which employs a receptor for the metabolic enzyme diamine oxidase (DAO) and results in cellular accumulation of the final metabolite methylimidazoleacetic acid. We demonstrate recognition and regulatory properties of DAO receptors as a function of cellular environmental conditions. Fast and slow ligand binding receptor populations bind DAO at 4 degrees C maximally in 1 and 7 h, respectively; upon warming cells to 37 degrees C both populations participate in degradative uptake of histamine accumulated as methylimidazoleacetic acid. Bound DAO is displaced by heparin with 24-fold greater potency than dextran sulfate, implicating structural specificity of heparin-like glycosaminoglycan moieties as a critical factor in initial receptor/enzyme interaction at fast and slow sites. Receptor-bound DAO is retained under mildly acidic conditions characteristic of early to mid endocytic intracellular compartments and thus could recycle to the plasma membrane intact after internalization. DAO initially bound to receptors in whole cells is retained through cell disruption/membrane fractionation procedures, but DAO binds poorly to isolated membrane fractions or presolubilized receptors, suggesting that the geometry of DAO binding components is not readily maintained upon cell disruption unless DAO is already bound. Cells down-regulate their complement of DAO receptors upon prolonged exposure to DAO. In cells plated at high density, half of the bound DAO becomes nondisplaceable by heparin within 15 min at 37 degrees C, a time consistent with receptor internalization, whereas cells plated at low density retain all bound DAO in a heparin-sensitive state. The protein kinase C activator phorbol 12-myristate 13-acetate modulates DAO receptor number by 35% and total histamine degradative uptake by > 2-fold. Thus this pathway is subject to regulation at the levels of DAO receptor numbers, their state of cell-surface display, and additional cellular elements of the degradative pathway with which the DAO receptors interface.

Urokinase binding and receptor identification in cultured endothelial cells.

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.

The histamine degradative uptake pathway in human vascular endothelial cells and skin fibroblasts is dependent on extracellular Na+ and Cl-.

We have previously reported that human vascular endothelial cells and skin fibroblasts carry out degradation of [3H]histamine by a mechanism involving two successive enzymatic steps: imidazole ring tele-methylation by the cells' endogenous methyltransferase and subsequent amine oxidation by an exogenous diamine oxidase. Both histamine and the exogenous second enzyme in the pathway associate with the cells via separate binding sites or receptors. The enzymatic degradation process results in cellular accumulation of the proximal and distal metabolites tele-methylhistamine and 1-methyl-4-imidazoleacetic acid (MIAA) (Haddock, R. C., Mack, P., Fogerty, F. J., and Baenziger, N. L. (1987) J. Biol. Chem. 262, 10220-10228). We have now demonstrated that this two-stage histamine degradative pathway is dependent on Na+ and Cl- in the extracellular environment. Accumulation of [3H] histamine-derived products is partially inhibited under conditions of Na+ deprivation and more substantially when Cl- is also withdrawn. The individual tele-methylation and amine oxidation enzymatic reactions themselves are unaffected or actually facilitated under these conditions. This indicates that it is the cellular mechanism for uptake coupled to the degradative pathway which reflects the cation and anion dependency. Restoration of degradative uptake displays a biphasic Na+ concentration curve, suggesting that the uptake process may be driven by multiple components. These findings indicate a role for both inward Na+ and Cl- ion movement in this cellular degradative uptake mechanism.

Role of receptors in metabolic interaction of histamine with human vascular endothelial cells and skin fibroblasts. An ordered sequence of enzyme action.

The interaction of histamine with an H1 receptor on human endothelial cells evokes production of the lipid mediator prostaglandin I2 (PGI2) and is accompanied by tachyphylaxis of this H1 receptor response (Baenziger, N. L., Fogerty, F. J., Mertz, L. F., and Chernuta, L. F. (1981) Cell 24, 915-923). We have explored the affected cells' capability for subsequent metabolic degradation of histamine molecules. Human vascular endothelial cells and skin fibroblasts exhibit a two-stage histamine degradation sequence whose participants are an enzyme native to the cells themselves and one provided from an extracellular source. Initially, the cells' endogenous histamine N-methyltransferase activity mediates conversion of cell-associated [3H]histamine to tele-methylhistamine with retention of this intermediate metabolite. Subsequently, in the presence of exogenous diamine oxidase derived from fetal calf serum or human placenta, cell-associated tele-methyl-histamine is further converted to the end product methylimidazoleacetic acid. After an initial lag phase lasting 3-6 min, the cell-associated radioactivity accumulates as methylimidazoleacetic acid at a linear rate substantially enhanced over that without diamine oxidase. The entire sequence is blocked by the histamine methyltransferase inhibitor homodimaprit. Accumulation of [3H]histamine metabolites by endothelial cells is saturable both with respect to exogenous diamine oxidase and to histamine. Thus this metabolic pathway is carried out at the level of the individual cell by means of binding sites or receptors for the substrate and for the distal degradative enzyme, diamine oxidase.