Biomedical subjects

D B Cines

Publications and source records attributed to D B Cines.

At least 55 records · Page 3Linked to original sources

PubMed1996

Unesterified long chain fatty acids inhibit the binding of single chain urokinase to the urokinase receptor.

The interaction of single chain urokinase with its receptor accelerates plasminogen activator activity on cell surfaces and induces intracellular signalling in several cell types. To date, no physiologic inhibitor of this binding has been identified. We report that the binding of scuPA to its cellular receptor is inhibited by long chain fatty acids such as oleic acid (C18, delta 9) at physiological plasma concentrations. Inhibition of single chain urokinase binding to human trophoblastic cells by long chain fatty acids was dose-dependent and saturable. Fifty percent of the binding was inhibited at an oleic acid concentration of 27 microM, while inhibition was maximal (75%) at 150 microM oleic acid. The inhibitory potency of oleic acid was unaffected by fatty acid free albumin or human plasma. Inhibition of single chain urokinase binding by free fatty acid analogues was critically dependent on chain length (> C14 required for inhibition) and was proportional to the extent of unsaturation. Only the fraction of specific scuPA binding to trophoblasts that was dependent on uPAR was susceptible to inhibition by oleic acid, while binding of scuPA to vitronectin, thombospondin, and the alpha 2-macroglobulin receptor/low-density lipoprotein-related receptor was not. [3H]Oleic acid bound specifically to recombinant soluble uPAR in a 1:1 molar ratio in the presence or absence of plasma and totally blocked its specific binding to a cell line expressing glycosyl phosphatidylinositol-linked single chain urokinase. These results indicate that oleic acid and other unsaturated long chain free fatty acids may serve as physiologic regulators of proteolytic events and intracellular signalling that depend upon the interaction of urokinase with its receptor.

Animals↗

PubMed1996

Antiplatelet antibody testing in thrombocytopenic pregnant women.

OBJECTIVE: The purpose of the study was to attempt to distinguish pregnant women with gestational thrombocytopenia from those with idiopathic immune thrombocytopenia by eight different platelet antibody assays. STUDY DESIGN: Sera from pregnant women with presumed gestational thrombocytopenia (n = 160) and idiopathic immune thrombocytopenia (n=90) were prospectively tested for indirect and platelet-associated immunoglobulins G and M and complement C3, as well as for serotonin release. After the results were analyzed, a subset of patients were subsequently analyzed for circulating antiplatelet antibody directed against platelet membrane glycoprotein GPIIb/IIIa. RESULTS: Indirect immunoglobulin G was significantly greater in the 85 women with idiopathic immune thrombocytopenia than in the 129 women with gestational thrombocytopenia (p<0.001). Platelet-associated immunoglobulin G was elevated in the majority of women, both those with gestational thrombocytopenia and those with idiopathic immune thrombocytopenia. There were also no statistically significant difference in the values for platelet-associated C3 or indirect immunoglobulin M and C3. Levels of platelet-associated immunoglobulin M showed a tendency to be higher in women with gestational thrombocytopenia (p=0.04), as did the values in the serotonin release assay (p=0.06). CONCLUSION: Our data demonstrate that patients with gestational thrombocytopenia had surprisingly high levels of platelet-associated immunoglobulin despite mild thrombocytopenia. Comparison of a relatively large number of patients with idiopathic immune thrombocytopenia and gestational thrombocytopenia indicates that women with idiopathic immune thrombocytopenia cannot be distinguished from those with gestational thrombocytopenia by means of one or more of the prototypic platelet antiglobulin tests currently in use. Our preliminary data with glycoprotein-specific assays indicate that they may be more useful.

Autoantibodies↗

PubMed1996

Thermotaxis of human trophoblastic cells.

The objective of this study was to determine whether cultured human trophoblasts migrate in response to changes in oxygen tension or temperature. Human trophoblastic cells distributed homogenously within individual wells of standard culture plates were subjected to oxygen and thermal gradients. The redistribution of cells was determined 90 min to 18 h after these gradients had been established. Trophoblastic cells did not migrate in response to gradients of oxygen or carbon dioxide applied in this manner. In contrast, the cells migrated in response to thermal gradients of less than 1 degree C in the direction of the warmer temperature. The response began within minutes, was reversed by a change in the direction of the thermal gradient, and was inhibited at high cell concentrations. Migration was independent of proliferation or protein synthesis, but required microfilament assembly. The capacity of trophoblasts to migrate in response to small difference of temperature within the physiologic range may contribute to the initiation of placental development before contact with the maternal circulation has been established.

Arteries↗

PubMed1995

Mapping the cell binding site on high molecular weight kininogen domain 5.

Investigations mapped the region(s) on the light chain of high molecular weight kininogen (HK) that participates in cell binding. Sequential and overlapping peptides of domain 5 (D5H) were synthesized to determine its cell binding site(s). Three peptides from non-overlapping regions on D5H were found to inhibit biotin-HK binding to endothelial cells. Peptides GKE19 and HNL 21 weakly inhibited biotin-HK binding with IC50 of 792 and 215 microM, respectively. Peptide HKH20 inhibited biotin-HK binding with an IC50 of 0.2 microM. Two peptides, GGH18 and HVL24, which overlapped HKH20, also inhibited biotin-HK binding to endothelial cells with IC50 values of 108 and 0.8 microM, respectively. Biotinylated HKH20 directly bound to endothelial cells. HK and HKH20 bound at or near the same site on endothelial cells because HK inhibited biotin-HKH20 binding (IC50 = 0.2 microM). A polyclonal anti-HKH20 antibody also blocked biotin-HK binding. Peptides HKH20 and HVL24 and anti-HKH20 antibody also inhibited the procoagulant activity of plasma HK. These data indicated that the cell and artificial surface binding sites on D5H overlap. The orientation of HK on endothelial cells may be critical for the assembly and activation of contact system enzymes and the expression of kininogen's anti-thrombin activity.

Amino Acid Sequence↗

PubMed1995

Enhancement of the enzymatic activity of single-chain urokinase plasminogen activator by soluble urokinase receptor.

Single-chain urokinase (scuPA), the unique form of urokinase secreted by cells, is converted to an active two-chain molecule through the cleavage of a single peptide bond by plasmin and other specific proteinases. Although scuPA may express limited enzymatic activity, its contribution to plasminogen activation on cell surfaces remains uncertain. Further, although it is well known that scuPA binds to a specific extracellular urokinase-type plasminogen activator receptor, the effect of this interaction on the enzymatic activity of scuPA has not been described. In the present paper we report that the binding of scuPA to cellular an to recombinant soluble urokinase-type plasminogen activator receptors (suPAR) increases its catalytic activity as measured by the cleavage of a urokinase-specific chromogenic substrate. suPAR increased the Vmax of scuPA 5-fold with little change in its Km. suPAR also stimulated the plasminogen activator activity of scuPA by decreasing its Km for Glu-plasminogen from 1.15 microM to 0.022 microM and by increasing the kcat of this reaction from 0.0015 to 0.022 s-1. Preincubation of scuPA with suPAR also enhances its susceptibility to inhibition by plasminogen activator inhibitor type 1, consistent with exposure of its catalytic site. The activity of scuPA bound to suPAR is not accompanied by cleavage of scuPA, which continues to migrate as a single band in SDS-polyacrylamide gel electrophoresis under reducing conditions. Moreover, suPAR increases the plasminogen activator activity of a plasmin-insensitive variant, scuPA (scuPA-Glu158), as well. Enhancement of scuPA activity by suPAR is both prevented and reversed by its aminoterminal fragment (amino acids 1-135), which competes for receptor binding, suggesting that continued binding to the receptor is required for expression of scuPA's enzymatic activity. Thus, our data suggest that scuPA may undergo a reversible transformation between a latent and an active state. The urokinase receptor may induce or stabilize scuPA in its active conformation, thereby contributing to the initiation of plasminogen activation on cell surfaces.

Peptide Fragments↗

PubMed1995

High-molecular-weight kininogen is exclusively membrane bound on endothelial cells to influence activation of vascular endothelium.

An important biologic function of high-molecular-weight kininogen (HK) is to deliver bradykinin (BK) to its cellular receptors. Internalization and degradation of HK may provide a mechanism by which endothelial cells modulate the production of BK and control its activities. Therefore, we investigated the binding and subsequent distribution of biotinylated-HK (biotin-HK) associated with human umbilical vein endothelial cells (HUVEC). HUVEC bound 3 to 4 times more HK and with greater avidity at 1 to 3 hours at 37 degrees C than at 4 degrees C (Bmax = 1.0 +/- 0.02 x 10(7) molecules/cell, kd = 7 +/- 3 nmol/L v Bmax = 2.6 +/- 0.2 x 10(6) molecules/cell, kd = 46 +/- 8 nmol/L). However, there was no evidence that the difference was caused by internalization of HK at the higher temperature. First, the same amount of biotin-HK was associated with nonpermeabilized and permeabilized HUVEC using buffers containing 20 to 50 mumol/L zinc ion in the absence or presence of 2 mmol/L calcium ion. Second, binding of biotin-HK to HUVEC was approximately 92% reversible at 1 hour when the cells were maintained at both 37 degrees C and 4 degrees C. Third, neither chloroquine nor primaquine altered the amount of biotin-HK bound to HUVEC. Fourth, biotin-HK bound to HUVEC was almost completely removed by pronase. Fifth, the nonpermeable dye, crystal violet, almost completely quenched the fluorescence signal emitted by HUVEC-associated fluorescein isothiocyanate (FITC) HK. Finally, the localization of HUVEC-bound FITC-HK was restricted to the membrane as shown by laser scanning confocal microscopy. The expression of HK binding sites had an absolute requirement for metabolic energy, but was not dependent on new protein synthesis. Membrane-bound HK contributed to the anticoagulant nature of endothelial cells by blocking human alpha-thrombin binding and its resultant induction of prostacyclin formation. These studies indicate that HK is not internalized by HUVEC, but remains primarily on cell surfaces to be accessible for BK liberation and to modulate the binding and actions of alpha-thrombin.

Blood Coagulation↗

PubMed1995

Factor IXa inhibition by protease nexin-2/amyloid beta-protein precursor on phospholipid vesicles and cell membranes.

Protease nexin-2/amyloid beta-protein precursor (PN-2/A beta PP) is a Kunitz-type protease inhibitor which has been shown to be a tight-binding inhibitor of enzymes, factors XIa and IXa (FIXa), suggesting a role for this protein in hemostasis. Since coagulant reactions are modulated on biologic surfaces, we investigated how 25:75 (mol/mol) phosphatidylserine/phosphatidylcholine vesicles (PSPC), thrombin-activated platelets, or umbilical vein endothelial cells influence inactivation of FIXa by PN-2/A beta PP. The Km of human or porcine FIXa activation of human factor X in the presence of PSPC, activated platelets, or endothelial cells in the absence or presence of thrombin-activated factor VIII (FVIIIa) was similar, (0.05-0.39 microM). The presence of FVIIIa increased the catalytic efficiency (kcat/Km ratio) of human and porcine factor IXa's activation of factor X 4952-406-fold, respectively. In the presence of PSPC, the Ki of human and porcine FIXa inhibition by PN-2/A beta PP was Ki = 1.9 x 10(-9) M and 5.8 x 10(-9) M, respectively. After the addition of FVIIIa to the reaction, the Ki for both human and porcine FIXa inhibition by PN-2/A beta PP on PSPC increased 13- and 4-fold to Ki = 2.5 x 10(-8) M and 2.4 x 10(-8) M, respectively. These Ki for inhibition of human FIXa on phospholipid vesicles by PN-2/A beta PP were similar when factor X activation was measured by chromogenic or activation peptide release assays. FVIIIa reduced the inhibition of FIXa by PN-2/A beta PP only in the presence of PSPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloid beta-Protein Precursor↗

PubMed1995

The antiphospholipid-protein syndrome.

The pathogenesis of the antiphospholipid syndrome remains uncertain. Antibodies that react with phospholipids may not be directly responsible for cellular injury, but may be part of the immune network through which autoantibodies with pathogenic potential are generated. The latter may recognize proteins such as beta 2-glycoprotein I that form complexes with phospholipids, proteins whose functions depend upon interaction with phospholipids such as protein C and its cofactors, altered lipoproteins such as oxidized low-density lipoproteins, or other molecules that share only antigenic similarity. Thus, a spectrum of autoantibodies that recognize different lipid-protein complexes may develop in these patients and contribute to the observed clinical heterogeneity of the syndrome. Current techniques do not permit identification of the subset of patients with antiphospholipid antibodies at risk for thrombosis or abortion and there are no prospective, controlled trials addressing the prophylaxis or treatment of affected individuals. Identification of the cellular targets of antibodies to lipid-protein moieties is needed to identify patients at risk for these complications and as a means to monitor therapy.

Abortion, Habitual↗

PubMed1995

Comparison of PF4/heparin ELISA assay with the 14C-serotonin release assay in the diagnosis of heparin-induced thrombocytopenia.

The diagnosis of heparin-induced thrombocytopenia (HIT) may be affirmed by demonstrating heparin-dependent anti-platelet antibodies using the 14C-serotonin release assay (SRA). In this study, results of the SRA was compared with the recently described platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). Compared with the SRA, the sensitivity and specificity of a PF4/heparin ELISA was 87% and 92%, respectively, using an assay developed in our laboratory; and 90% and 98%, respectively, using a commercially developed kit (Diagnostica Stago, Asnieres, France). However, antibodies to PF4/heparin were also detected in up to 8% of patients whose plasma was negative by SRA, and 23% of patients receiving heparin who were not thrombocytopenic. These data indicate that results obtained with the PF4/heparin ELISA and the SRA are generally in accord in patients with a clinical diagnosis of HIT. However, discrepant results occur in approximately 20% of cases because of the greater sensitivity of ELISA and the possible involvement of other heparin-binding proteins. The fact that each assay contributes independent information in some cases must be considered in the sequence of test performance and in providing consultation to the practicing hematologist.

Adult↗

PubMed1994

The carboxyl terminus of bradykinin and amino terminus of the light chain of kininogens comprise an endothelial cell binding domain.

Bradykinin (BK), a potent vasoactive nonapeptide formed by proteolytic cleavage of kininogen, mediates its activity by binding to specific cell surface receptors. Delivery of BK to these receptors is limited by cell-bound and plasma kininases that degrade BK to inactive fragments. Binding of its parent compound, kininogen, to specific endothelial cell receptors may provide an environment in which the degradation of BK by kininases is restricted. The determinants that mediate kininogen binding to endothelial cells have not been fully elucidated. The present studies suggest that part of BK and the amino-terminal amino acids of kininogens' common light chain constitute part of this recognition sequence. Human umbilical vein endothelial cells (HUVEC) in culture at 37 degrees C express 2-3-fold more binding sites for biotinylated high molecular weight kininogen (biotin-HK) containing BK than for biotin-HK from which BK has been liberated by plasma kallikrein. Binding of BK-free biotin-HK was not restored by preincubating HUVEC with BK, arguing against the possibility that BK released from biotin-HK stimulated expression of additional HK receptors. The extent of biotin-HK binding to HUVEC at 37 degrees C directly correlated with the amount of BK retained within the protein. Four lines of evidence suggest that part of BK and the amino terminus of the light chain of kininogens are part of the sequence recognized by the endothelial cell kininogen receptor. First, monoclonal antibodies to the carboxyl terminus of BK (MBK3) and the common light chains of the kininogens (HKL6, HKL8) inhibited biotin-HK binding to HUVEC. Second, a synthetic, dimeric bradykinin receptor antagonist blocked biotin-HK (IC50 = 9 microM) binding to HUVEC as did two synthetic tissue kallikrein inhibitors modeled after the carboxyl-terminal sequence of BK. Third, synthetic peptides containing the carboxyl-terminal portion of BK and the amino terminus of kininogens' common light chain, MKRPPGFSPFRSSRIG and GFSPFRSSRIG, blocked binding of biotin-HK (IC50 = 2-3 mM), whereas an overlapping peptide, SPFRSSRIGEIKEETT, at 5 mM and a scrambled peptide, FSGPKRSPIMGRPSFR, did not. Fourth, biotinylated GMKRPPGFSPFRSSRIG specifically bound to HUVEC, and its binding was blocked by HK. Since the presence of the nonapeptide BK in HK contributes to maximal binding of HK to HUVEC, there is a novel type of BK receptor in which part of the nonapeptide is recognized within the context of its parent molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Endocytosis of urokinase-plasminogen activator inhibitor type 1 complexes bound to a chimeric transmembrane urokinase receptor.

The urokinase receptor (uPAR) is linked to plasma membranes through a glycosylphosphatidylinositol (GPI) anchor. It has been posited that the GPI anchor facilitates clearance of uPAR-bound complexes between two chain urokinase (tcuPA) and plasminogen activator inhibitor type 1 (PAI-1) by the alpha 2-macroglobulin receptor (alpha 2MR) which permits re-expression of unoccupied uPA receptors on the cell surface. To test this hypothesis we compared internalization and degradation of 125I-labeled tcuPA-PAI-1 by COS cells expressing either transfected wild-type, GPI-linked uPAR (uPAR/GPI), or a chimeric receptor composed of the extracellular domains of uPAR linked to the transmembrane and cytosolic domains of the alpha chain (p55 subunit) of the interleukin-2 receptor (uPAR/IL-2R alpha). The kinetics of binding, internalization and degradation of tcuPA-PAI-1 by COS cells expressing each form of uPAR were virtually identical. However, internalization of complexes by uPAR/IL-2R alpha was more susceptible to inhibition by recombinant soluble 39-kDa alpha 2MR-associated protein (RAP) which competes for binding of tcuPA-PAI-1 complexes to alpha 2MR (p < 0.001), and the internalization was accompanied by a greater reduction in the number of surface uPAR/IL-2R alpha, than uPAR/GPI (p < 0.05). These studies indicate that the rate of internalization of tcuPA-PAI-1 is governed primarily by the extracellular domains of uPAR, whereas the GPI anchor may facilitate internalization of complexes and re-expression of uPAR when binding sites on alpha 2MR are limiting.

Animals↗

PubMed1994

Expression of urokinase receptors by human trophoblast. A histochemical and ultrastructural analysis.

BACKGROUND: Through their ability to invade endometrium, remodel the uterine spiral arteries, and sustain placental blood fluidity, trophoblast cells play a central role in establishing and maintaining the integrity of the uteroplacental vasculature. The expression of urokinase receptors by trophoblast may facilitate these processes by focusing plasminogen activator activity to discrete sites on the cell surface and promoting the activation of cell-bound plasminogen. However, although urokinase receptors are expressed by cultured trophoblast, the expression of these receptors by trophoblast in vivo has not been examined. EXPERIMENTAL DESIGN: Immunohistochemistry and immunoelectron microscopy were used to characterize the expression of urokinase receptors by villous and extravillous trophoblast at several points in gestation. RESULTS: Urokinase receptors were expressed in a polarized fashion at the leading edge of migrating extravillous trophoblast cells. Receptors were also abundantly expressed during the first and second trimesters of gestation by villous trophoblast, where they were located on apical villous projections and within intracellular vacuoles, a subset of which were lysosomes. CONCLUSIONS: The polarized expression of urokinase receptors by invasive extravillous trophoblast cells is consistent with a role for these receptors in mediating the extent and directionality of trophoblast migration. In contrast, the expression of urokinase receptors by villous trophoblast, which are not actively invasive in vivo, may serve to facilitate the generation of plasmin at the interface of these cells with maternal plasma, thereby limiting the deposition of fibrin within the placental intervillous spaces. Diminished urokinase receptor expression by villous trophoblast at term may represent a physiologic adaptation to diminish local fibrinolysis and limit hemorrhage at parturition.

Cell Movement↗

PubMed1993

Detection of antitrophoblast antibodies in the sera of patients with anticardiolipin antibodies and fetal loss.

Women with anticardiolipin antibodies (ACLA) are at increased risk for fetal loss. One potential explanation for this outcome is that sera from these individuals contain antibodies reactive with trophoblast cells, which are involved in the establishment of the uteroplacental vasculature and maintenance of placental blood fluidity. To examine this hypothesis, we compared the incidence of trophoblast-reactive antibodies in 27 patients with ACLA and a history of fetal loss with that in 29 normal pregnant women. Sera from 20 patients, but only one control, contained trophoblast-reactive antibodies (P < .001). These antibodies were not directed against major histocompatibility class I antigens, and reacted with both term and first-trimester trophoblast cells. In most cases, sera from which ACLA were adsorbed by cardiolipin-containing liposomes maintained reactivity against cells. In addition, patient Ig fractions immunoprecipitated an approximately 62-kD protein from the trophoblast cell surface, stimulated the release of arachidonic acid and thromboxane A2 by trophoblasts, and inhibited the binding of prourokinase to trophoblast urokinase receptors. These observations show that sera from women with ACLA and a history of fetal loss contain antitrophoblast antibodies. These antibodies may be serologically distinct from ACLA, and may contribute to the pathogenesis of fetal demise.

Abortion, Spontaneous↗

PubMed1993

Overexpression of urokinase receptor increases matrix invasion without altering cell migration in a human osteosarcoma cell line.

Proteolysis triggered by receptor-bound urokinase-type plasminogen activator (uPA) involves a cascade of species-specific molecular interactions. To study the role of the uPA receptor (uPAR) in such interactions, a human osteosarcoma cell line (HOS), which normally expresses low levels of uPAR, was transfected with human uPAR complementary DNA. One of several stably transformed clonal cells lines, designated 2A2, was characterized and compared to the parental HOS, revealing the following: (a) stable incorporation of uPAR complementary DNA into the genome demonstrated by Southern blot analysis; (b) a 10-fold increase in steady state mRNA levels of uPAR assessed by Northern blot analysis; (c) a 2-fold increase in the surface expression of glycosylphosphatidylinositol anchored uPAR protein determined by enzyme-linked immunosorbent assay and by the specific binding of radiolabeled single chain uPA; (d) a 2-fold increase in internalization and degradation of radiolabeled uPA/PAI-1 complexes; and (e) a 2-fold increase in receptor-bound uPA-mediated plasmin generation measured by the cleavage of a chromogenic substrate and degradation of 125I-labeled laminin. The involvement of uPAR in cellular processes was determined by comparing 2A2 and HOS cells in in vitro migration and invasion assays. The migration of 2A2 cells were slower on fibronectin-coated surfaces in a linear under-agarose assay, but both cell lines migrated at the same rate on uncoated polycarbonate filters in Boyden chamber assays. In the invasion experiments, 4 times more 2A2 than HOS cells penetrated through the barrier of reconstituted basement membrane Matrigel. These data suggest that uPAR does not potentiate random cell migration but facilitates matrix degradation and subsequent cell invasion.

Base Sequence↗

PubMed1993

Determinants of binding and internalization of tissue-type plasminogen activator by human vascular smooth muscle and endothelial cells.

Vascular injury induced by angioplasty is often followed by smooth muscle cell (SMC) proliferation, migration, and accumulation of extracellular matrix. Since plasminogen activators and their receptors may be important both in cell migration and the clearance of plasminogen activators, we studied the binding, internalization, and degradation of radiolabeled tissue-type plasminogen activator (t-PA) by explant cultures of human vascular SMC. Binding of t-PA to SMC at 4 degrees C was rapid, specific, saturable, and inhibitable by antibodies to plasminogen activator inhibitor type 1 (PAI-1). At 37 degrees C, labeled t-PA was internalized and degraded by SMC but not by human umbilical vein endothelial cells. Internalization and degradation was mediated by the low density lipoprotein receptor related protein/alpha 2-macroglobulin receptor (LRP) in that these processes were inhibited by an anti-LRP antibody, recombinant LRP-associated protein, urokinase-type plasminogen activator-PAI-1 complexes, and lactoferrin. The portion of t-PA most important for internalization after complexing with PAI-1 is likely to be in the finger and/or epidermal growth factor domains or in the carbohydrate at amino acid 117, in that the internalization of preformed t-PA.PAI-1 complexes or complexes formed on the cell surface was inhibited by an excess of active site-blocked wild type t-PA, but not by an active site blocked t-PA variant missing these domains. These studies are consistent with a model in which t-PA binds initially to SMC-associated PAI-1 with subsequent t-PA.PAI-1 internalization via LRP. SMC may play an important role in clearing t-PA-PAI-1 complexes from within the vessel wall.

Binding Sites↗

PubMed1993

Bradykinin regulates the expression of kininogen binding sites on endothelial cells.

The vasoactive compound bradykinin (BK) is liberated by proteolytic cleavage from high molecular weight kininogen (HK) and low molecular weight kininogen (LK). Expression of kininogens on cell surface receptors may affect the delivery of BK at sites of inflammation. Therefore, we investigated whether BK itself alters the expression of binding sites for its parent molecules, HK and LK, on the surface of cultured human umbilical vein endothelial cells (HUVEC). 125I-LK and 125I-HK each bind to a single class of sites on HUVEC in reactions that are saturable, reversible, and zinc-dependent (Bmax = 9.7 +/- 0.2 x 10(5) sites/cell; kd = 43.3 +/- 8 nmol/L; n = 5 and Bmax = 10.3 +/- 0.4 x 10(5) sites/cell; kd = 40.3 +/- 0.9 nmol/L; n = 3 for LK and HK, respectively). HK and LK compete for the same binding site (Ki = 19.4 +/- 5 nmol/L HK v 125I-LK; Ki = 24.5 +/- 4 nmol/L LK v 125I-HK, n = 3). Moreover, 50-fold molar excess light chain of HK inhibits 125I-LK binding 51% and 50-fold molar excess LK and the heavy chain of HK inhibit 125I-light chain of HK binding 92% and 76%, respectively. Preincubation of HUVEC with BK produces a transient, concentration-dependent increase in the binding of HK and LK, reaching a maximum 3 to 4 hours after addition of BK (46% increase over control for HK; 57% increase over control for LK; P < .005 for each ligand). Des-Arg9-bradykinin, a B1 receptor agonist, increases kininogen binding to the same extent as BK; the upregulation of kininogen binding sites by BK is partially blocked by a B1 but not by a B2 receptor antagonist. The protein kinase C inhibitors (PKC), sphingosine and H7, completely block the induction of HK receptors by BK. Phorbol 12-myristate 13-acetate (PMA), which also activates PKC, stimulates the binding of HK and the purified light chain of HK to HUVEC as well. However, unlike HK and its light chain, binding of LK and the heavy chain of HK are increased by PMA only in the presence of added calcium ion. These studies show that BK upregulates a common binding site for HK, LK, and each chain of HK on HUVEC. Induction of kininogen receptors on endothelial cells by BK may modulate the generation of this vasoactive compound at sites of vascular injury.

Bradykinin↗

PubMed1993

Plasma concentration of endothelin-1 in women with cocaine-associated pregnancy complications.

OBJECTIVE: The purpose of this study was to determine if the plasma concentration of endothelin-1 is elevated in pregnant women abusing cocaine and to determine how these levels differ from those in patients with preeclampsia and in women with uncomplicated pregnancies. STUDY DESIGN: Plasma endothelin-1 levels were measured in 30 women with acute cocaine intoxication, 32 women with preeclampsia, 14 pregnant women with chronic hypertension, 26 women with uncomplicated pregnancies, and 16 nonpregnant individuals. Serial samples after delivery were obtained in 12 women with preeclampsia, 10 with cocaine abuse, 4 with chronic hypertension, and 7 with uncomplicated pregnancies. RESULTS: The mean endothelin-1 concentration in those with cocaine abuse was 18.2 +/- 8.1 pg/ml (95% confidence interval 15.2 to 21.2). This was similar to that in women with preeclampsia (21.1 +/- 5.9 pg/ml, 95% confidence interval 19 to 23.3) (p = 0.2) but significantly different from that in women with chronic hypertension (11.5 +/- 3.6 pg/ml, 95% confidence interval 9.4 to 13.6) (p < 0.001) and women with uncomplicated pregnancies (6.7 +/- 3.9 pg/ml, 95% confidence interval 5.1 to 8.2) (p < 0.001). CONCLUSIONS: Endothelin-1 levels in women abusing cocaine are comparable to those in women with preeclampsia and are significantly higher than those in gravid women with chronic hypertension and women with uncomplicated pregnancies. Elevated levels of endothelin-1 may contribute to some of the pregnancy-related complications in women abusing cocaine.

Adult↗

PubMed1993

Regulation of the endothelial cell urokinase-type plasminogen activator receptor. Evidence for cyclic AMP-dependent and protein kinase C-dependent pathways.

Binding of urokinase-type plasminogen activator (u-PA) to specific receptors (u-PAR) on the surface of endothelial cells contributes to the regulation of plasmin-dependent processes such as fibrinolysis and angiogenesis. We studied the effect of raising intracellular levels of cyclic AMP (cAMP) and/or activating protein kinase C on the expression of u-PAR in cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with forskolin stimulated a time- and concentration-dependent increase in the expression of u-PAR, measured both by an increase in the specific binding of radiolabeled single-chain u-PA (scu-PA) and by increased binding of anti-u-PAR antibodies. Maximal increase in u-PAR expression (81 +/- 11% above control, n = 11) was not associated with a change in receptor affinity for scu-PA when HUVEC were incubated for 20 hours at 37 degrees C with 50 microM forskolin. Receptor induction by forskolin was inhibited when HUVEC were preincubated with deoxyadenosine monophosphate (DAM), an inhibitor of adenylyl cyclase. A similar increase in receptor expression (128 +/- 27% above control, n = 3) was induced by the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate (50 mM). Forskolin induced an approximately twofold increase in the expression of a single approximately 1.4-kb u-PAR messenger RNA (mRNA) transcript within 2 hours. Phorbol myristate acetate (PMA) also stimulated a time- and concentration-dependent increase in specific scu-PA binding. The maximal increase in u-PAR expression (254 +/- 27% above control, n = 11) was observed when HUVEC were preincubated with 10 nM PMA for 20 hours. Induction of u-PAR by PMA was inhibited when HUVEC were preincubated with either cycloheximide or H7 but was unaffected by DAM. u-PAR induced by PMA showed a reduced affinity for scu-PA (Kd, 14 +/- 2 nM versus 3.6 +/- 0.6 nM, p < 0.001; n = 8). PMA stimulation for 20 hours resulted in a sixfold increase in a single approximately 1.4-kb u-PAR mRNA transcript, with increased levels detectable within 30 minutes. Coincubation of HUVEC with optimal concentrations of forskolin and PMA for 20 hours produced a fully additive increase in u-PAR expression at both the mRNA and protein levels. These data suggest that both cAMP-dependent and protein kinase C-dependent protein kinase pathways may independently regulate u-PAR expression in human endothelial cells.

Blotting, Northern↗