We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of (125)I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol. min(-1). 10(6) cells(-1). Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-beta-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased (125)I-albumin uptake 2.3-fold. At 37 degrees C, (125)I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC(50) = 1 microM). Using a two-site model, we estimated by Scatchard analysis the affinity (K(D)) and maximal capacity (B(max)) of albumin uptake to be 0.87 microM (K(D1)) and 0.47 pmol/10(6) cells (B(max1)) and 93.3 microM (K(D2)) and 20.2 pmol/10(6) cells (B(max2)). At 4 degrees C, we also observed two populations of specific binding sites, with high (K(D1) = 13.5 nM, 1% of the total) and low (K(D2) = 1.6 microM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.
We investigated the function of proteinase-activated receptor-1 (PAR-1) in the regulation of pulmonary microvascular permeability in response to thrombin challenge using PAR-1 knockout mice (-/-). Lungs were isolated and perfused with albumin (5 g/100 ml)-Krebs solution at constant flow (2 ml/min). Lung wet weight and pulmonary artery pressure (P(pa)) were continuously monitored. We determined the capillary filtration coefficient (K(fc)) and (125)I-labeled albumin (BSA) permeability-surface area product (PS) to assess changes in pulmonary microvessel permeability to liquid and albumin, respectively. Normal and PAR-1-null lung preparations received in the perfusate: 1) thrombin or 2) selective PAR-1 agonist peptide (TFLLRNPNDK-NH(2)). In control PAR-1 (+/+) mouse lungs, (125)I-albumin PS and K(fc) were significantly increased over baseline (by approximately 7- and 1.5-fold, respectively) within 20 min of alpha-thrombin (100 nM) challenge. PAR-1 agonist peptide (5 microM) gave similar results, whereas control peptide (5 microM; FTLLRNPNDK-NH(2)) was ineffective. At relatively high concentrations, thrombin (500 nM) or PAR-1 agonist peptide (10 microM) also induced increases in P(pa) and lung wet weight. All effects of thrombin (100 or 500 nM) or PAR-1 agonist peptide (5 or 10 microM) were prevented in PAR-1-null lung preparations. Baseline measures of microvessel permeability and P(pa) in the PAR-1-null preparations were indistinguishable from those in normal lungs. Moreover, PAR-1-null preparations gave normal vasoconstrictor response to thromboxane analog, U-46619 (100 nM). The results indicate that the PAR-1 receptor is critical in mediating the permeability-increasing and vasoconstrictor effects of thrombin in pulmonary microvessels.
Abstract-Caveolin-1, the caveolae scaffolding protein, binds to and negatively regulates eNOS activity. As caveolin-1 also regulates caveolae-mediated endocytosis after activation of the 60-kDa albumin-binding glycoprotein gp60 in endothelial cells, we addressed the possibility that endothelial NO synthase (eNOS)-dependent NO production was functionally coupled to caveolae internalization. We observed that gp60-induced activation of endocytosis increased NO production within 2 minutes and up to 20 minutes. NOS inhibitor N G -nitro-L-arginine (L-NNA) prevented the NO production. To determine the role of caveolae internalization in the mechanism of NO production, we expressed dominant-negative dynamin-2 mutant (K44A) or treated cells with methyl--cyclodextrin. Both interventions inhibited caveolae-mediated endocytosis and NO generation induced by gp60. We determined the role of signaling via Src kinase in the observed coupling of endocytosis to eNOS activation. Src activation induced the phosphorylation of caveolin-1, Akt and eNOS, and promoted dissociation of eNOS from caveolin-1. Inhibitors of Src kinase and Akt also prevented NO production. In isolated perfused mouse lungs, gp60 activation induced NO-dependent vasodilation, whereas the response was attenuated in eNOS Ϫ/Ϫ or caveolin-1 Ϫ/Ϫ lungs. Together, these results demonstrate a critical role of caveolae-mediated endocytosis in regulating eNOS activation in endothelial cells and thereby the NO-dependent vasomotor tone. Key Words: caveolin-1 Ⅲ endocytosis Ⅲ vasomotor tone Ⅲ albumin Ⅲ transcytosis E ndothelial NO synthase (eNOS) is modified by N-myristoylation and palmitoylation, which targets the enzyme to caveolae, 1 the plasma membrane cholesterol-rich microdomains. 2,3 Multiple mechanisms are involved in regulating NO production following eNOS activation. eNOS activity is regulated by Ca 2ϩ -calmodulin, phosphorylation activated by kinases such as Src and interactions with caveolin-1, dynamin, and heat shock protein 90 (HSP90). 4 The effects of phosphorylation are complex. Phosphorylation of Ser116 and Thr497 negatively regulates eNOS activity, whereas phosphorylation at Ser635 and Ser1179 has the opposite effect. 5-7 Src kinase activated eNOS by inducing phosphorylation at Tyr83. 8 Phosphorylation at Ser617 functions by affecting the phosphorylation of the above residues. 9 Insulin, estrogen, and shear stress were shown to induce phosphorylationdependent activation of eNOS at Ser1179 independent of increased intracellular [Ca 2ϩ ] 10 -12 eNOS in caveolae is held inactive by its association with caveolin-1, 13 but eNOS activity can be increased by Ca 2ϩ /calmodulin 3 and binding to HSP90 and dynamin-2. 14,15 HSP90 facilitates the phosphorylation of eNOS by forming a ternary complex with eNOS and Akt. 16 Dynamin-2 regulates eNOS activity through the binding of its proline-rich domain to the FAD domain of eNOS, promoting electron transfer between the bound flavins of the reductase domain and increasing NO production. 15 We have shown that activation of t...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.