Evidence for endothelium-derived relaxing factor in cultured cells

Intracellular cyclic GMP concentration was used as a biochemical indicator of endothelium-dependent and organonitrate-induced responses to these vasodilators in cultured porcine aortic smooth muscle and endothelial cells. Sodium nitroprusside (10(-6) M) caused a rapid increase in cyclic GMP levels in confluent smooth muscle cell cultures but not in confluent endothelial monolayers. Adenosine triphosphate (10(-4) M) and methacholine (10(-5) M), two agents that elicit endothelium-dependent relaxation in intact vessels, failed to raise cyclic GMP concentrations in muscle or endothelial cultures alone. When the cell types were grown together in mixed culture, however, treatment with adenosine triphosphate or methacholine induced an elevation in intracellular cyclic GMP levels. These findings suggest that mixed cultures of arterial smooth muscle and endothelial cells can be used to study the phenomenon of endothelium-dependent responses in arterial smooth muscle.     

Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis

To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters before and after exposure to xanthine and xanthine oxidase. Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion. Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments. The oxidant- induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 hours in tissue culture media enriched with fetal bovine serum. However, incubation in tissue culture media without serum resulted in progressive injury and cell death. Hence, the brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum. Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis. Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells. The calcium antagonist lanthanum chloride prevented xanthine- xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. These data suggest that oxygen radicals can reversibly increase endothelial permeability to macromolecules, that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial calcium homeostasis.     

D1 dopamine receptor signaling involves caveolin-2 in HEK-293 cells

BACKGROUND: Dopamine receptors in the kidney, especially those belonging to the D1-like receptor family, are important in the regulation of renal function and blood pressure. Because of increasing evidence that G protein-coupled receptors (GPCRs) are associated with caveolae and lipid rafts, we tested the hypothesis that the D1 dopamine receptor (D1R) and signaling molecules are regulated by caveolin in caveolae or lipid rafts. METHODS: Six experimental approaches were used: (1) construction of tagged human D1Rs (hD1Rs) and transfectants; (2) cell culture [human embryonic kidney (HEK)-293 and immortalized rat renal proximal tubule cells] and biotinylation; (3) cell fractionation by sucrose gradient centrifugation; (4) immunoprecipitation and immunoblotting; (5) immunofluorescence and confocal microscopy; and (6) adenylyl cyclase assays. RESULTS: hD1Rs, heterologously expressed in HEK-293 cells, formed protein species with molecular mass ranging from 50 to 250 kD, and were localized in lipid rafts and nonraft plasma membranes. The hD1Rs cofractionated with caveolin-2, G protein subunits, and several signaling molecules. Both exogenously expressed hD1Rs and endogenously expressed rat D1Rs colocalized and coimmunoprecipitated with caveolin-2. A D1R agonist (fenoldopam) increased the amount of caveolin-2beta associated with hD1Rs and activated adenylyl cyclase to a greater extent in lipid rafts than in nonraft plasma membranes. Reduction in the expression of caveolin-2 with antisense oligonucleotides attenuated the stimulatory effect of fenoldopam on cyclic adenosine monophosphate (cAMP) accumulation. CONCLUSION: The majority of hD1Rs are distributed in lipid rafts. Heterologously and endogenously expressed D1Rs in renal cells are associated with and regulated by caveolin-2.