Since thiols can undergo nitrosation and the cell membrane is rich in thiol-containing proteins, we considered the possibility that membrane surface thiols may regulate cellular entry of NO. Recently, protein disulfide isomerase (PDI), a protein that catalyzes thio–disulfide exchange reactions, has been found on the cell-surface membrane. We hypothesized that cell-surface PDI reacts with NO, catalyzes S-nitrosation reactions, and facilitates NO transfer from the extracellular to intracellular compartment. We observed that PDI catalyzes the S-nitrosothiol–dependent oxidation of the heme group of myoglobin (15-fold increase in the rate of oxidation compared with control), and that NO reduces the activity of PDI by 73.1 ± 21.8% (P < 0.005). To assess the role of PDI in the cellular action of NO, we inhibited human erythroleukemia (HEL) cell-surface PDI expression using an antisense phosphorothioate oligodeoxynucleotide directed against PDI mRNA. This oligodeoxynucleotide decreased cell-surface PDI content by 74.1 ± 9.3% and PDI folding activity by 46.6 ± 3.5% compared with untreated or “scrambled” phosphorothioate oligodeoxynucleotide–treated cells (P < 0.0001). This decrease in cell-surface PDI was associated with a significant decrease in cyclic guanosine monophosphate (cGMP) generation after S-nitrosothiol exposure (65.4 ± 26.7% reduction compared with control; P < 0.05), with no effect on cyclic adenosine monophosphate (cAMP) generation after prostaglandin E1 exposure. These data demonstrate that the cellular entry of NO involves a transnitrosation mechanism catalyzed by cell-surface PDI. These observations suggest a unique mechanism by which extracellular NO gains access to the intracellular environment.
Adrian Zai, M. Audrey Rudd, Anne Ward Scribner, Joseph Loscalzo
Estrogen is an important vasoprotective molecule that causes the rapid dilation of blood vessels by activating endothelial nitric oxide synthase (eNOS) through an unknown mechanism. In studies of intact ovine endothelial cells, 17β-estradiol (E2) caused acute (five-minute) activation of eNOS that was unaffected by actinomycin D but was fully inhibited by concomitant acute treatment with specific estrogen receptor (ER) antagonists. Overexpression of the known transcription factor ERα led to marked enhancement of the acute response to E2, and this was blocked by ER antagonists, was specific to E2, and required the ERα hormone-binding domain. In addition, the acute response of eNOS to E2 was reconstituted in COS-7 cells cotransfected with wild-type ERα and eNOS, but not by transfection with eNOS alone. Furthermore, the inhibition of tyrosine kinases or mitogen-activated protein (MAP) kinase kinase prevented the activation of eNOS by E2, and E2 caused rapid ER-dependent activation of MAP kinase. These findings demonstrate that the short-term effects of estrogen central to cardiovascular physiology are mediated by ERα functioning in a novel, nongenomic manner to activate eNOS via MAP kinase–dependent mechanisms.
Zhong Chen, Ivan S. Yuhanna, Zoya Galcheva-Gargova, Richard H. Karas, Michael E. Mendelsohn, Philip W. Shaul
Human granulocytic ehrlichiosis (HGE) is an emerging tickborne illness caused by an intracellular bacterium that infects neutrophils. Cells susceptible to HGE express sialylated Lewis x (CD15s), a ligand for cell selectins. We demonstrate that adhesion of HGE to both HL60 cells and normal bone marrow cells directly correlates with their CD15s expression. HGE infection of HL60 cells, bone marrow progenitors, granulocytes, and monocytes was blocked by monoclonal antibodies against CD15s. However, these antibodies did not inhibit HGE binding, and anti-CD15s was capable of inhibiting the growth of HGE after its entry into the target cell. In contrast, neuraminidase treatment of HL60 cells prevented both HGE binding and infection. A cloned cell line (HL60-A2), derived from HL60 cells and resistant to HGE, was deficient in the expression of α-(1,3)fucosyltransferase (Fuc-TVII), an enzyme known to be required for CD15s biosynthesis. Less than 1% of HL60-A2 cells expressed CD15s, and only these rare CD15s-expressing cells bound HGE and became infected. After transfection with Fuc-TVII, cells regained CD15s expression, as well as their ability to bind HGE and become infected. Thus, CD15s expression is highly correlated with susceptibility to HGE, and it, and/or a closely related sialylated and α-(1,3) fucosylated molecule, plays a key role in HGE infection, an observation that may help explain the organism's tropism for leukocytes.
Jesse L. Goodman, Curtis M. Nelson, Marina B. Klein, Stanley F. Hayes, Brent W. Weston
We investigated the possible involvement of the autonomic nervous system in the effect of a long-term elevation of plasma free fatty acid (FFA) concentration on glucose-induced insulin secretion (GIIS) in rats. Rats were infused with an emulsion of triglycerides (Intralipid) for 48 hours (IL rats). This resulted in a twofold increase in plasma FFA concentration. At the end of infusion, GIIS as reflected in the insulinogenic index (ΔI/ΔG) was 2.5-fold greater in IL rats compared with control saline-infused rats. The ratio of sympathetic to parasympathetic nervous activities was sharply decreased in IL rats relative to controls. GIIS was studied in the presence of increasing amounts of α- and β-adrenoreceptor agonists and antagonists. The lowest concentrations of the α2A-adrenoreceptor agonist oxymetazoline, which were ineffective in control rats, reduced GIIS in IL rats. At the dose of 0.3 pmol/kg, GIIS became similar in IL and control rats. The use of β-adrenoreceptor agonist (isoproterenol) or antagonist (propranolol) did not result in a significant alteration in GIIS in both groups. GIIS remained as high in IL vagotomized rats as in intact IL rats, indicating that changes in parasympathetic tone were of minor importance. Altogether, the data show that lipid infusion provokes β-cell hyperresponsiveness in vivo, at least in part through changes in α2-adrenergic innervation.
Christophe Magnan, Stephan Collins, Marie-France Berthault, Nadim Kassis, Mylène Vincent, Marc Gilbert, Luc Pénicaud, Alain Ktorza, Françoise Assimacopoulos-Jeannet
Evidence for increased oxidant stress has been reported in human atherosclerosis. However, no information is available about the importance of in situ oxidant stress in relation to plaque stability. This information is relevant because the morbidity and mortality of atherosclerosis are essentially the consequences of acute ischemic syndromes due to unstable plaques. We studied 30 carotid atherosclerotic plaques retrieved by endarterectomy from 18 asymptomatic (stable plaques) and 12 symptomatic patients (unstable plaques). Four normal arteries served as controls. After lipid extraction and ester hydrolysis, quantitation of different indices of oxidant stress were analyzed, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatetraenoic acids (EETs), ketoeicosatetraenoic acids (oxo-ETEs), and F2-isoprostanes using online reverse-phase high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS). All measurements were carried out in a strictly double-blind procedure. We found elevated levels of the different compounds in atherosclerotic plaques. Levels of HETEs were 24 times higher than EETs, oxo-ETEs, or F2-isoprostanes. Levels of HETEs, but not those of EETs, oxo-ETEs or F2-isoprostanes, were significantly elevated in plaques retrieved from symptomatic patients compared with those retrieved from asymptomatic patients (1,738 ± 274 vs. 1,002 ± 107 pmol/μmol lipid phosphorous, respectively; P < 0.01). One monooxygenated arachidonate species, 9-HETE, which cannot be derived from known enzymatic reactions, was the most abundant and significant compound observed in plaques, suggesting that nonenzymatic lipid peroxidation predominates in advanced atherosclerosis and may promote plaque instability.
Ziad Mallat, Tatsuji Nakamura, Jeanny Ohan, Guy Lesèche, Alain Tedgui, Jacques Maclouf, Robert C. Murphy
In the absence of exogenous glucocorticoids, decreasing media pH (from 7.4 to 6.8) for 24 hours increased the Na+/H+ exchanger 3 (NHE3) activity in opossum kidney (OKP) cells. 10–7 M and 10–8 M hydrocortisone increased NHE3 activity, and in their presence, acid incubation further increased NHE3 activity. Hydrocortisone (10–9 M) had no effect on NHE3 activity, but in its presence, the effect of acid incubation on NHE3 activity increased twofold. Aldosterone (10–8 M) had no effect. In the absence of hydrocortisone, acid incubation increased NHE3 protein abundance by 47%; in the presence of 10–9 M hydrocortisone, acid incubation increased NHE3 protein abundance by 132%. The increase in NHE3 protein abundance was dependent on protein synthesis. However, 10–9 M hydrocortisone did not modify the effect of acid incubation to cause a twofold increase in NHE3 mRNA abundance. In the absence of protein synthesis, 10–9 M hydrocortisone did potentiate an effect of acid on NHE3 activity, which was due to trafficking of NHE3 to the apical membrane. These results suggest that glucocorticoids and acid interact synergistically at the level of NHE3 translation and trafficking.
Patrice M. Ambühl, Xiaojing Yang, Yan Peng, Patricia A. Preisig, Orson W. Moe, Robert J. Alpern
Features that distinguish tumor vasculatures from normal blood vessels are sought to enable the destruction of preformed tumor vessels. We show that blood vessels in both a xenografted tumor and primary human tumors contain a sizable fraction of immature blood vessels that have not yet recruited periendothelial cells. These immature vessels are selectively obliterated as a consequence of vascular endothelial growth factor (VEGF) withdrawal. In a xenografted glioma, the selective vulnerability of immature vessels to VEGF loss was demonstrated by downregulating VEGF transgene expression using a tetracycline-regulated expression system. In human prostate cancer, the constitutive production of VEGF by the glandular epithelium was suppressed as a consequence of androgen-ablation therapy. VEGF loss led, in turn, to selective apoptosis of endothelial cells in vessels devoid of periendothelial cells. These results suggest that the unique dependence on VEGF of blood vessels lacking periendothelial cells can be exploited to reduce an existing tumor vasculature.
Laura E. Benjamin, Dragan Golijanin, Ahuva Itin, Dov Pode, Eli Keshet
We examined potential mechanisms by which angiotensin subtype-2 (AT2) receptor stimulation induces net fluid absorption and serosal guanosine cyclic 3′,5′-monophosphate (cGMP) formation in the rat jejunum. l-arginine (l-ARG) given intravenously or interstitially enhanced net fluid absorption and cGMP formation, which were completely blocked by the nitric oxide (NO) synthase inhibitor, N-nitro-l-arginine methylester (l-NAME), but not by the specific AT2 receptor antagonist, PD-123319 (PD). Dietary sodium restriction also increased jejunal interstitial fluid cGMP and fluid absorption. Both could be blocked by PD or l-NAME, suggesting that the effects of sodium restriction occur via ANG II at the AT2 receptor. l-ARG–stimulated fluid absorption was blocked by the soluble guanylyl cyclase inhibitor 1-H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (ODQ). Cyclic GMP–specific phosphodiesterase in the interstitial space decreased extracellular cGMP content and prevented the absorptive effects of l-ARG. Angiotensin II (ANG II) caused an increase in net Na+ and Cl– ion absorption and 22Na+ unidirectional efflux (absorption) from the jejunal loop. In contrast, intraluminal heat-stable enterotoxin of Escherichia coli (STa) increased loop cGMP and fluid secretion that were not blocked by either l-NAME or ODQ. These findings suggest that ANG II acts at the serosal side via AT2 receptors to stimulate cGMP production via soluble guanylyl cyclase activation and absorption through the generation of NO, but that mucosal STa activation of particulate guanylyl cyclase causes secretion independently of NO, thus demonstrating the opposite effects of cGMP in the mucosal and serosal compartments of the jejunum.
Xiao-Hong Jin, Helmy M. Siragy, Richard L. Guerrant, Robert M. Carey
Allergic asthma, which is present in as many as 10% of individuals in industrialized nations, is characterized by chronic airway inflammation and hyperreactivity induced by allergen-specific Th2 cells secreting interleukin-4 (IL-4) and IL-5. Because Th1 cells antagonize Th2 cell functions, it has been proposed that immune deviation toward Th1 can protect against asthma and allergies. Using an adoptive transfer system, we assessed the roles of Th1, Th2, and Th0 cells in a mouse model of asthma and examined the capacity of Th1 cells to counterbalance the proasthmatic effects of Th2 cells. Th1, Th2, and Th0 lines were generated from ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice and transferred into lymphocyte-deficient, OVA-treated severe combined immunodeficiency (SCID) mice. OVA-specific Th2 and Th0 cells induced significant airway hyperreactivity and inflammation. Surprisingly, Th1 cells did not attenuate Th2 cell–induced airway hyperreactivity and inflammation in either SCID mice or in OVA-immunized immunocompetent BALB/c mice, but rather caused severe airway inflammation. These results indicate that antigen-specific Th1 cells may not protect or prevent Th2-mediated allergic disease, but rather may cause acute lung pathology. These findings have significant implications with regard to current therapeutic goals in asthma and allergy and suggest that conversion of Th2-dominated allergic inflammatory responses into Th1-dominated responses may lead to further problems.
Gesine Hansen, Gerald Berry, Rosemarie H. DeKruyff, Dale T. Umetsu
Hyperglycemia can cause vascular dysfunctions by multiple factors including hyperosmolarity, oxidant formation, and protein kinase C (PKC) activation. We have characterized the effect of hyperglycemia on p38 mitogen-activated protein (p38) kinase activation, which can be induced by oxidants, hyperosmolarity, and proinflammatory cytokines, leading to apoptosis, cell growth, and gene regulation. Glucose at 16.5 mM increased p38 kinase activity in a time-dependent manner compared with 5.5 mM in rat aortic smooth muscle cells (SMC). Mannitol activated p38 kinase only at or greater than 22 mM. High glucose levels and a PKC agonist activated p38 kinase, and a PKC inhibitor, GF109203X, prevented its activation. However, p38 kinase activation by mannitol or tumor necrosis factor-α was not inhibited by GF109203X. Changes in PKC isoform distribution after exposure to 16.5 mM glucose in SMC suggested that both PKC-β2 and PKC-δ isoforms were increased. Activities of p38 kinase in PKC-δ– but not PKC-β1–overexpressed SMC were increased compared with control cells. Activation of p38 kinase was also observed and characterized in various vascular cells in culture and aorta from diabetic rats. Thus, moderate hyperglycemia can activate p38 kinase by a PKC-δ isoform–dependent pathway, but glucose at extremely elevated levels can also activate p38 kinase by hyperosmolarity via a PKC-independent pathway.
Masahiko Igarashi, Hisao Wakasaki, Noriko Takahara, Hidehiro Ishii, Zhen-Y Jiang, Teruaki Yamauchi, Koji Kuboki, Matthias Meier, Christopher J. Rhodes, George L. King
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