Modulation of endothelial NO production by high-density lipoprotein.
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Animals↗
Biomedical subjects
C Mineo
Publications and source records attributed to C Mineo.
Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i).
Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.
Animals↗
Potocytosis. Robert Feulgen Lecture.
Potocytosis represents a mechanism by which small and large molecules as well as macromolecular complexes are sequestered and transported by caveolae. Caveolae are flask-shaped plasma membrane specializations characterized by a filamentous coat consisting of caveolins that decorates the inside surface of each caveola membrane. They have endocytotic functions that differ from the clathrin-coated pit pathway. Ligands bound to receptors that are internalized by caveolae can be delivered to four different locations in the cell bypassing the lysosome and at least four different caveolae membrane traffic patterns during potocytosis can be distinguished. Hence, cells have two endocytic machines and each is designed to accomplish different tasks. This review provides a brief summary of the discovery of caveolae and of potocytosis, and focuses on recent discoveries of the unique endocytic capabilities of caveolae in a variety of different cells.
Animals↗
Estrogen receptor alpha and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae.
Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) alpha-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ERalpha and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E(2), 10(-8) mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ERalpha antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ERalpha displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ERalpha plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ERalpha protein in caveolae, and E(2) caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E(2) on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ERalpha is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org.
Acetylcholine↗
Synaptotagmin regulation of coated pit assembly.
Synaptotagmins bind clathrin AP-2 with high affinity via their second C(2) domain, which indicates they are involved in coated pit function. We now report that expression of synaptotagmins lacking either the second C(2) domain or the entire cytoplasmic region potently inhibit endocytosis. Inhibition was dependent on two intramembrane cysteine residues that were found to be essential for synaptotagmin oligomerization. Cells expressing the wild-type, but not the mutant, truncated synaptotagmin fragment had a reduced number of clathrin-coated pits. These results suggest that the formation of synaptotagmin multimers is an important step in the regulation of coated pit assembly.
Adaptor Protein Complex alpha Subunits↗
Regulated migration of epidermal growth factor receptor from caveolae.
In quiescent fibroblasts, epidermal growth factor (EGF) receptors (EGFR) are initially concentrated in caveolae but rapidly move out of this membrane domain in response to EGF. To better understand the dynamic localization of EGFR to caveolae, we have studied the behavior of wild-type and mutant receptors expressed in cells lacking endogenous EGFR. All of the receptors we examined, including those missing the first 274 amino acids or most of the cytoplasmic tail, were constitutively concentrated in caveolae. By contrast, migration from caveolae required EGF binding, an active receptor kinase domain, and at least one of the five tyrosine residues present in the regulatory domain of the receptor. Movement appears to be modulated by Src kinase, is blocked by activators of protein kinase C, and occurs independently of internalization by clathrin-coated pits. Two mutant receptors previously shown to induce an oncogenic phenotype lack the ability to move from caveolae in response to EGF, suggesting that a prolonged residence in this domain may contribute to abnormal cell behavior.
Amino Acid Substitution↗
Polarized distribution of endogenous Rac1 and RhoA at the cell surface.
Rac1 and RhoA regulate membrane ruffling and stress fiber formation. Both molecules appear to exert their control from the plasma membrane. In fibroblasts stimulated with platelet-derived growth factor or lysophosphatidic acid, the reorganization of the cytoskeleton begins at specific sites on the cell surface. We now report that endogenous Rac1 and RhoA also have a polarized distribution at the cell surface. Cell fractionation and immunogold labeling show that in quiescent fibroblasts both of these molecules are concentrated in caveolae, which are plasma membrane domains that are associated with actin-rich regions of the cell. Treatment of these cells with platelet-derived growth factor stimulated the recruitment of additional Rac1 and RhoA to caveolae fractions, while lysophosphatidic acid only caused the recruitment of RhoA. We could reconstitute the recruitment of RhoA using either whole cell lysates or purified caveolae. Surprisingly, pretreatment of the lysates with exoenzyme C3 shifted both resident and recruited RhoA from caveolae to noncaveolae membranes. The shift in location was not caused by inactivation of the RhoA effector domain. Moreover, chimeric proteins containing the C-terminal consensus site for Rac1 and RhoA prenylation were constitutively targeted to caveolae fractions. These results suggest that the polarized distribution of Rho family proteins at the cell surface involves an initial targeting of the protein to caveolae and a mechanism for retaining it at this site.
Amino Acid Sequence↗
Targeting of protein kinase Calpha to caveolae.
Previously, we showed caveolae contain a population of protein kinase Calpha (PKCalpha) that appears to regulate membrane invagination. We now report that multiple PKC isoenzymes are enriched in caveolae of unstimulated fibroblasts. To understand the mechanism of PKC targeting, we prepared caveolae lacking PKCalpha and measured the interaction of recombinant PKCalpha with these membranes. PKCalpha bound with high affinity and specificity to caveolae membranes. Binding was calcium dependent, did not require the addition of factors that activate the enzyme, and involved the regulatory domain of the molecule. A 68-kD PKCalpha-binding protein identified as sdr (serum deprivation response) was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKCalpha binding. A 100-amino acid sequence from the middle of sdr competitively blocked PKCalpha binding while flanking sequences were inactive. Caveolae appear to be a membrane site where PKC enzymes are organized to carry out essential regulatory functions as well as to modulate signal transduction at the cell surface.
Amino Acid Sequence↗
Physical association with ras enhances activation of membrane-bound raf (RafCAAX).
The transforming activity of artificially membrane-targeted Raf1 suggests that Ras-mediated recruitment of Raf1 to the plasma membrane is an important step in Raf1 activation. Cellular Ras is concentrated in the caveolae, a microdomain of the plasma membrane that is highly enriched in caveolin, glycosylphosphatidylinositol-anchored proteins, and signal transduction molecules. Growth factor stimulation recruits Raf1 to this membrane domain. Whether Ras simply promotes Raf1 association with caveolae membranes or also modulates subsequent activation events is presently unclear. We have identified a ras variant, ras12V,37G, that does not interact with Raf1 but does interact with a mutant raf1, raf1(257L). To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX, membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V, 37G. Cell fractionation localized both raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of ras12V,37G expression; however, coexpression of ras12V,37G enhanced the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by induction of cellular transformation, increased Raf kinase activity, and induction of activated MAP kinase. These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from membrane recruitment.
3T3 Cells↗
Localization of epidermal growth factor-stimulated Ras/Raf-1 interaction to caveolae membrane.
An essential step in the epidermal growth factor (EGF)-dependent activation of MAP kinase is the recruitment of Raf-1 to the plasma membrane. Here we present evidence that caveolae are the membrane site where Raf-1 is recruited. Caveolae fractions prepared from normal Rat-1 cells grown in the absence of serum were highly enriched in both EGF receptors and Ras. Thirty seconds after EGF was added to these cells Raf-1 began to appear in caveolae but not in non-caveolae membrane fractions. The maximum concentration was reached at 3 min followed by a decline over the next 60 min. During this time EGF receptors disappeared from the caveolae fraction while the concentration of Ras remained constant. The Raf-1 in this fraction was able to phosphorylate MAP kinase kinase, whereas cytoplasmic Raf-1 in the same cell was inactive. Elevation of cellular cAMP blocked the recruitment of Raf-1 to caveolae. Overexpression of Ha-RasV12 caused the recruitment of Raf-1 to caveolae independently of EGF stimulation, and this was blocked by the farnesyltransferase inhibitor BZA-5B. Finally, prenylation appeared to be required for localization of Ras to caveolae.
Animals↗
A vacuolar-type proton ATPase mediates acidification of plasmalemmal vesicles during potocytosis.
Previously we showed that the potocytosis of 5-methyltetrahydrofolate is dependent on the acidification of plasmalemmal vesicles created each time a caveolae, closes off from the cell surface. We now report that bafilomycin A1, which is a specific inhibitor of the V-type proton ATPase, inhibits 5-methyltetrahydrofolate uptake into MA104 cells (ED50 = 150 nM). The inhibitory effect was reversed within 30 min after removal of the drug from the cells. Bafilomycin A1 had no effect on the binding of folic acid to its receptor. A concentration of up to 200 nM bafilomycin A1 did not affect sequestration of folate receptors. Immunoblotting showed that the 70-kDa subunit of the V-type proton pump was localized to caveolae-rich fractions isolated from the plasma membrane of these cells. These results suggest that a V-type proton pump acidifies the lumen of plasmalemmal vesicles during potocytosis.
Acids↗
Exocrine function of caerulein-induced acute pancreatitis in anesthetized rats.
Exocrine function was studied in anesthetized rats that had received two specific doses of caerulein (maximal stimulation and supramaximal stimulation). Male Wistar rats (body weight, 200-250 g) were divided into three groups: the control group (4-h saline infusion), the maximal stimulation group (0.25 microgram/kg per h caerulein for 4 h), and the caerulein pancreatitis group (10 micrograms/kg per h for 4h). Histologically, interstitial edema and cytoplasmic vacuolization were observed only in the caerulein pancreatitis group, with no abnormal findings in the other groups. The volume of pancreatic juice was significantly increased in both the maximal stimulation group and the caerulein pancreatitis group. The protein output and the amylase output in the 1st h of caerulein infusion were also significantly increased, to 459% and 338% in the maximal stimulation group, and to 925% and 1430% respectively, in the caerulein pancreatitis compared to the baseline values. We also found that the pancreatic juice of the caerulein pancreatitis group contained precipitated protein, and high trypsin activity, and protein degradation was confirmed by electrophoresis. These findings were not observed in the other groups. These results strongly suggest that hypersecretion and the appearance of trypsin activity in pancreatic juice plays an important role in the induction of histological changes in this pancreatitis model in anesthetized rats.
Acute Disease↗
Clustered folate receptors deliver 5-methyltetrahydrofolate to cytoplasm of MA104 cells.
Previously, a high affinity, glycosylphosphatidylinositol-anchored receptor for folate and a caveolae internalization cycle have been found necessary for potocytosis of 5-methyltetrahydrofolate in MA104. We now show by cell fractionation that folate receptors also must be clustered in caveolae for potocytosis. An enriched fraction of caveolae from control cells retained 65-70% of the [3H]folic acid bound to cells in culture. Exposure of cells to the cholesterol-binding drug, filipin, which is known to uncluster receptors, shifted approximately 50% of the bound [3H]folic acid from the caveolae fraction to the noncaveolae membrane fraction and markedly inhibited internalization of [3H]folic acid. An mAb directed against the folate receptor also shifted approximately 50% of the caveolae-associated [3H]folic acid to noncaveolae membrane, indicating the antibody perturbs the normal receptor distribution. Concordantly, the mAb inhibited the delivery of 5-methyl[3H]tetrahydrofolate to the cytoplasm. Receptor bound 5-methyl[3H]tetrahydrofolate moved directly from caveolae to the cytoplasm and was not blocked by phenylarsine oxide, an inhibitor of receptor-mediated endocytosis. These results suggest cell fractionation can be used to study the uptake of molecules by caveolae.
Animals↗
A detergent-free method for purifying caveolae membrane from tissue culture cells.
Current methods for purifying caveolae from tissue culture cells take advantage of the Triton X-100 insolubility of this membrane domain. To circumvent the use of detergents, we have developed a method that depends upon the unique buoyant density of caveolae membrane. The caveolae fractions that we obtain are highly enriched in caveolin. As a consequence we are able to identify caveolae-associated proteins that had previously gone undetected. Moreover, resident caveolae proteins that are soluble in Triton X-100 are retained during the isolation.
Biomarkers↗
Angiotensin II binding activity in cultured porcine arterial endothelial cells.
Angiotensin II (A II) binding activity was detected in the particulate fraction (100,000 g, 60 min precipitate) of cultured porcine aortic endothelial cells. Scatchard analysis of the binding activity indicated a single class of binding sites with a dissociation constant (Kd) of 1.1 nM and a total binding capacity (Bmax) of 125 fmol/mg protein. The binding of [125I]A II was inhibited by excess unlabelled A II, A II analogues ([Sar1, Ile8]A II and [Sar1, Ala8]A II), A I (angiotensin I) and A III (angiotensin III), but not by bradykinin. Type specific A II receptor antagonists, losartan (type 1 angiotensin II receptor) and PD123319 (type 2 angiotensin II receptor), did not inhibit the binding. These results suggest that the A II specific binding protein(s) or receptor(s) is present in arterial endothelial cells, and that it is different from typical type 1 and type 2 angiotensin II receptors.
Angiotensin II↗
Intracellular action of an exogenous low-molecular-weight synthetic protease inhibitor, E3123, in cerulein-induced acute pancreatitis in rats.
The intracellular distribution and action of a new synthetic protease inhibitor, E3123, were studied in cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced by a 4-h iv infusion of a supramaximal dose of cerulein, and was treated by prophylactic (pretreatment) or therapeutic (posttreatment) continuous administration of E3123. Pancreatic edema and hyperamylasemia were ameriolated only by prophylactic treatment. A subcellular fractionation study showed that the activities of cathepsin-B and trypsin in the zymogen granule-enriched fraction of the cerulein-pancreatitis group were remarkably increased. Both prophylactic and therapeutic treatment significantly prevented the elevation of these enzyme activities. These effects were accompanied by amelioration of pancreatic histopathological features, including intracellular vacuolization and fat necrosis. A microscopic autoradiographic study using 3H-labeled E3123 showed diffuse intracellular distribution of E3123, and the radioactivity of 3H-E3123 in the posttreatment group was three times greater than that in the pretreatment group. This study provides the first experimental evidence that, even when administered therapeutically, exogenous protease inhibitors are transported into pancreatic acinar cells, thereby reducing the severity of early intracellular alterations in cerulein-induced acute pancreatitis.
Acute Disease↗
Isolation of a cDNA encoding thymic shared antigen-1. A new member of the Ly6 family with a possible role in T cell development.
We have previously characterized a novel mouse thymocyte marker, defined as thymic shared Ag-1 (TSA-1), present on both immature thymocytes and a subset of thymic medullary epithelial cells. MTS 35, a mAb specific for TSA-1, alters T cell differentiation when added to fetal thymic organ cultures, suggesting TSA-1 may be important for T cell development in the thymus. In this study, we describe the isolation of a cDNA encoding TSA-1 using transient expression of COS-7 cells and selection with MTS 35. The predicted amino acid sequence of this cDNA encodes a 15 to 17-kDa protein and the expressed protein is linked to the membrane via a phosphatidylinositol moiety. TSA-1 is transcriptionally active at various levels in all organs examined, suggesting that its role is not solely intrathymic. TSA-1 shares amino acid sequence homology to the mouse Ly6 multigene family, epidermal growth factor-like receptors, and to cobra venom neurotoxin. The Tsa-1 locus is located on chromosome 15 linked to Ly6 on the mouse genome. We also examined the effects of MTS 35 in fetal thymic organ cultures repopulated with two subsets of thymocytes representing defined stages of T cell development. Our results suggest that TSA-1 may play a role during positive selection and the transition from CD4+CD8+ thymocytes to the mature CD4+CD8- and CD4-CD8+ subsets.
Amino Acid Sequence↗
Intercellular transport through a partially denuded arterial endothelial monolayer. Effect of platelets and PGI2.
Fluorescein Dextran (FD) was shown to be transported at increased rates through partially denuded endothelial monolayer. Platelet binding to the partially denuded monolayer lowered transport rates to those comparable with intact endothelium. Inhibition of transport by platelet binding was not affected by the addition of isocarbacyclin (a stable derivative of PGI2). This result suggests that adherent platelets at the partial denudation site are sufficient to suppress transport of FD.
Animals↗