Biomedical subjects

F Murad

Publications and source records attributed to F Murad.

At least 19 recordsLinked to original sources

PubMed2001

YC-1 activation of human soluble guanylyl cyclase has both heme-dependent and heme-independent components.

YC-1 [3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole] is an allosteric activator of soluble guanylyl cyclase (sGC). YC-1 increases the catalytic rate of the enzyme and sensitizes the enzyme toward its gaseous activators nitric oxide or carbon monoxide. In other studies the administration of YC-1 to experimental animals resulted in the inhibition of the platelet-rich thrombosis and a decrease of the mean arterial pressure, which correlated with increased cGMP levels. However, details of YC-1 interaction with sGC and enzyme activation are incomplete. Although evidence in the literature indicates that YC-1 activation of sGC is strictly heme-dependent, this report presents evidence for both heme-dependent and heme-independent activation of sGC by YC-1. The oxidation of the sGC heme by 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one completely inhibited the response to NO, but only partially attenuated activation by YC-1. We also observed activation by YC-1 of a mutant sGC, which lacks heme. These findings indicate that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme.

Base Sequence↗

PubMed2001

Diversity of endotoxin-induced nitrotyrosine formation in macrophage-endothelium-rich organs.

The administration of bacterial lipopolysaccharide (LPS; endotoxin) can stimulate the development of the systemic inflammatory response syndrome, which can compromise the function of many organ systems, resulting in multiple organ failure. Activation of macrophages and cytokines by endotoxin and the subsequent formation of reactive oxygen and nitrogen species are of central pathogenic importance in various inflammatory diseases including sepsis. However, whether different tissues behave the same in pathological changes produced by LPS and what factors may affect pathological processes and protein tyrosine nitration in different organs, still remain to be evaluated. In the present study, we investigated the distribution of nitrotyrosine and other pathological changes induced by LPS in rat liver, spleen, and lung, all of which are rich in macrophages and endothelial cells. Our study revealed two important findings: first, a denitration activity in spleen white pulp might play a key role to protect the areas from nitration. Similar activity might also exist in endothelial cells of sinusoids and capillaries. Second, protein nitration might not induce significant tissue damage as shown in liver and spleen. However, inflammatory infiltration with increased formation NO* and other reactive species may result in severe tissue injury, as demonstrated in lung after LPS administration.

Animals↗

PubMed2001

Nitration of succinyl-CoA:3-oxoacid CoA-transferase in rats after endotoxin administration.

The tyrosine nitration of proteins has been observed in diverse inflammatory conditions and has been linked to the presence of reactive nitrogen species. From many in vitro experiments, it is apparent that tyrosine nitration may alter the function of proteins. A limited number of experiments under in vivo conditions also demonstrate that protein nitration is associated with altered cellular processes. To understand the association of protein nitration with the pathogenic mechanism of the disease, it is essential to identify specific protein targets of nitration with in vivo or intact tissue models. Using anti-nitrotyrosine antibodies, we demonstrated the accumulation of nitrotyrosine in a 52-kDa protein in rat kidney after lipopolysaccharide treatment. The 52-kDa protein was purified and identified with partial sequence as succinyl-CoA:3-oxoacid CoA-transferase (SCOT; EC ). Western blot analysis revealed that the nitration of this mitochondrial enzyme increased in the kidneys and hearts of lipopolysaccharide-treated rats, whereas its catalytic activity decreased. These data suggest that tyrosine nitration may be a mechanism for the inhibition of SCOT activity in inflammatory conditions. SCOT is a key enzyme for ketone body utilization. Thus, tyrosine nitration of the enzyme with sepsis or inflammation may explain the altered metabolism of ketone bodies present in these disorders.

Amino Acid Sequence↗

PubMed2001

Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus.

Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

Animals↗

PubMed2001

Organ sites of lipopolysaccharide-induced nitric oxide production in the anesthetized rat.

The objective of this research was to determine the amount and timing of nitric oxide (NO, nitrogen monoxide) gas produced by the lungs, intestinal mucosa, and organ surfaces facing the peritoneal cavity after iv injection of a bacterial toxin, lipopolysaccharide (LPS). Some of the deleterious effects of LPS on organ function have been attributed to NO or strong oxidants formed locally from NO. Medical-grade air was used as an inspiratory air source (50 strokes/min x 3 ml/stroke) or was pumped through the ileal lumen or peritoneal cavity (20 strokes/min x 3 ml/stroke). The air was collected at intervals of 15-30 min for 3 h after LPS and analyzed for authentic NO gas by chemiluminescence. LPS (5 mg/kg) or saline was injected iv. Sodium nitroprusside (SNP) was injected to determine the appearance of its NO released into the perfused compartments. Blood pressure, plasma nitrate plus nitrite (NO(x)), and total plasma leukocytes were measured as other manifestations of LPS effects. NO began to increase in the pulmonary expired air 90 min after LPS and continued to increase for the remainder of the experiment. The final pulmonary post-LPS [NO] was about 20-fold greater than the [NO] before LPS. LPS had no effect on intraluminal or intraperitoneal [NO]. The saline injection had no effect on [NO] in any compartment. SNP injection increased NO entry into all three air-perfused compartments. Thus, NO from an exogenous tissue source was not prevented from being detected. Blood pressure was decreased by LPS only during the pulmonary perfusion. There were no significant effects of LPS on leukocytes or plasma NO(x). LPS decreased blood pressure and leukocytes and increased plasma NO(x) when air perfusion was not done. It was concluded that different organs can produce LPS-induced NO at markedly different rates and times. However, some aspect of the experimental technique of air perfusion could alter the effects of LPS.

Anesthesia, General↗

PubMed2001

Expression of nitric oxide synthase isoforms and nitrotyrosine immunoreactivity by B-cell non-Hodgkin's lymphomas and multiple myeloma.

AIMS: Nitric oxide synthases (NOS) are isoenzymes that catalyse the synthesis of nitric oxide (NO). The three main NOS isoforms are: NOS1 or neuronal, NOS2 or inducible, and NOS3 or endothelial. NO plays both physiological and pathological roles, depending on its rate of synthesis and concentration, cellular source and microenvironment. Apoptosis is an important biological factor in low-grade lymphomas, and NO is able to prevent apoptosis. In-situ expression of NOS and synthesis of NO have been shown in several malignant tumours, but not in lymphoid neoplasms. This study evaluates whether human B-cell neoplasms express NOS isoforms, and nitrotyrosine (NY), which is usually interpreted as a marker of NO. METHODS AND RESULTS: We studied the expression of NOS-IR isoforms and NY-IR in 16 cases of B-cell non-Hodgkin's lymphoma (NHL) (five follicle centre cell lymphoma, four small lymphocytic/CLL, and seven diffuse large cell lymphoma), and 10 cases of multiple myeloma (MM). NOS1 was expressed in 5/10 cases of MM, and 15/16 cases of NHL. NOS2 was detected in all cases of MM, and in 14/16 cases of NHL, whereas NOS3 was positive in 3/10 of MM and in only in 1/16 cases of NHL. The expression of NY-IR was observed in 70% of MM cases, and in all cases of B-cell NHL, in a dot-like pattern in few tumour cells. CONCLUSIONS: B-cell neoplasms express neuronal and inducible NOS, and nitrotyrosine. Taken together, our results suggest that B-cell neoplasms can produce NO. The role of NO in the biology, diagnosis and prognosis of B-cell neoplasms remains to be established.

Adult↗

PubMed2001

Down-regulation of inducible nitric-oxide synthase (NOS-2) during parasite-induced gut inflammation: a path to identify a selective NOS-2 inhibitor.

Nitric oxide (NO) possesses potent anti-inflammatory properties; however, an over-production of NO will promote inflammation and induce cell and tissue dysfunction. Thus, the ability to precisely regulate NO production could prove beneficial in controlling damage. In this study, advantage was taken of the well characterized inflammatory response caused by an intestinal parasite, Trichinella spiralis, to study the relationship between intestinal inflammation and the regulation of nitric oxide synthase-type 2 (NOS-2) expression. Our study revealed that a specific gut inflammatory reaction results in inhibition of NOS-2 expression. Characteristics of this inhibition are: 1) local jejunal inflammation induced by T. spiralis systemically inhibits NOS-2 gene transcription, protein expression, and enzyme activity; 2) the inhibition blunts endotoxin-stimulated NOS-2 expression; 3) the inhibition does not extend to the expression of other isoforms of NOS, to paxillin, a housekeeper protein, or to cyclooxygenase-2, another protein induced by proinflammatory cytokines; 4) the inhibition is unlikely related to the formation of specific anti-parasite antibodies; and 5) the inhibition may involve substances other than stress-induced corticosteroids. Elucidation of such potent endogenous NOS-2 down-regulatory mechanisms could lead to the development of new strategies for the therapy of inflammatory conditions characterized by the overproduction of NO.

Animals↗

PubMed2001

Novel effects of nitric oxide.

Nitric oxide (NO), a simple free radical gas, elicits a surprisingly wide range of physiological and pathophysiological effects. NO interacts with soluble guanylate cyclase to evoke many of these effects. However, NO can also interact with molecular oxygen and superoxide radicals to produce reactive nitrogen species that can modify a number of macromolecules including proteins, lipids, and nucleic acids. NO can also interact directly with transition metals. Here, we have reviewed the non--3',5'-cyclic-guanosine-monophosphate-mediated effects of NO including modifications of proteins, lipids, and nucleic acids.

Animals↗

PubMed2001

Diabetes-associated nitration of tyrosine and inactivation of succinyl-CoA:3-oxoacid CoA-transferase.

High levels of reactive species of nitrogen and oxygen in diabetes may cause modifications of proteins. Recently, an increase in protein tyrosine nitration was found in several diabetic tissues. To understand whether protein tyrosine nitration is the cause or the result of the associated diabetic complications, it is essential to identify specific proteins vulnerable to nitration with in vivo models of diabetes. In the present study, we have demonstrated that succinyl-CoA:3-oxoacid CoA-transferase (SCOT; EC 2.8.3.5) is susceptible to tyrosine nitration in hearts from streptozotocin-treated rats. After 4 and 8 wk of streptozotocin administration and diabetes progression, SCOT from rat hearts had a 24% and 39% decrease in catalytic activity, respectively. The decrease in SCOT catalytic activity is accompanied by an accumulation of nitrotyrosine in SCOT protein. SCOT is a mitochondrial matrix protein responsible for ketone body utilization. Ketone bodies provide an alternative source of energy during periods of glucose deficiency. Because diabetes results in profound derangements in myocardial substrate utilization, we suggest that SCOT tyrosine nitration is a contributing factor to this impairment in the diabetic heart.

Animals↗

PubMed2000

Cutting edge: targeted disruption of the C3a receptor gene demonstrates a novel protective anti-inflammatory role for C3a in endotoxin-shock.

The complement anaphylatoxin C3a, on binding the C3aR, mediates numerous proinflammatory activities. In addition, recent in vitro studies with C3a have implicated C3aR as a possible anti-inflammatory receptor. Because of its possible dual role in modulating the inflammatory response, it is uncertain whether C3aR contributes to the pathogenesis of endotoxin shock. Here, the targeted-disruption of the C3aR in mice is reported. These mice exhibit an enhanced lethality to endotoxin shock with a pronounced gene dosage effect. In addition, the plasma concentration of IL-1beta was significantly elevated in the C3aR(-/-) mice compared with their littermates following LPS challenge. These findings demonstrate an important protective role for the C3aR in endotoxin shock and indicate that, in addition to its traditionally accepted functions in mediating inflammation, the C3aR also acts in vivo as an anti-inflammatory receptor by attenuating LPS-induced proinflammatory cytokine production.

Amino Acid Sequence↗

PubMed2000

Molecular cloning of a regulatory protein for membrane-bound guanylate cyclase GC-A.

Activation of membrane-bound guanylate cyclase GC-A by atrial natriuretic factor (ANF) may require the involvement of accessory proteins. To identify these postulated proteins, we isolated a 1. 0-kb cDNA clone from a rat brain expression library using a polyclonal antiserum against mastoparan. The 1.0-kb cDNA encodes a protein of 111 amino acids. Expression of this cDNA in COS-7 cells potentiated ANF-stimulated GC-A activity. Therefore, the 1.0-kb gene encodes a guanylate cyclase regulatory protein (GCRP). Fluorescence microscopy studies using the fusion protein of GCRP with green fluorescence protein (GFP) indicated that GCRP was present in the cytosol in PC12 cells, but translocated toward the plasma membrane in the presence of ANF. Coimmunoprecipitation experiments indicate that GCRP associates with GC-A in the presence of ANF. These results suggest that ANF induces the association of GCRP with GC-A and this association contributes to the activation of GC-A.

Amino Acid Sequence↗

PubMed2000

Genomic organization of alpha1 and beta1 subunits of the mammalian soluble guanylyl cyclase genes.

The structures of the genes encoding the alpha(1) and beta(1) subunits of murine soluble guanylyl cyclase (sGC) were determined. Full-length cDNAs isolated from mouse lungs encoding the alpha(1) (2.5 kb) and beta(1) (3.3 kb) subunits are presented in this report. The alpha(1) sGC gene is approximately 26.4 kb and contains nine exons, whereas the beta(1) sGC gene spans 22 kb and consists of 14 exons. The positions of exon/intron boundaries and the sizes of introns for both genes are described. Comparison of mouse genomic organization with the Human Genome Database predicted the exon/intron boundaries of the human genes and revealed that human and mouse alpha1 and beta1 sGC genes have similar structures. Both mouse genes are localized on the third chromosome, band 3E3-F1, and are separated by a fragment that is 2% of the chromosomal length. The 5' untranscribed regions of alpha(1) and beta(1) subunit genes were subcloned into luciferase reporter constructs, and the functional analysis of promoter activity was performed in murine neuroblastoma N1E-115 cells. Our results indicate that the 5' untranscribed regions for both genes possess independent promoter activities and, together with the data on chromosomal localization, suggest independent regulation of both genes.

Animals↗

PubMed2000

Human recombinant soluble guanylyl cyclase: expression, purification, and regulation.

The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

Guanylate Cyclase↗

PubMed2000

Cellular signaling with nitric oxide and cyclic guanosine monophosphate.

The understanding of the formation and biological actions of nitric oxide (NO) has grown extensively during the past two decades. With the discoveries of the biological effects of NO and nitrovasodilators on cyclic guanosine monophosphate, with the elucidation of the biochemical mechanisms of NO synthesis, and with the growing knowledge of regulation of NO synthases, the complexities of this signal transduction cascade and its participation in numerous cell signaling processes continues. NO can be recognized as an intracellular second messenger, a local substance for regulation of neighboring cells, a neurotransmitter, and probably a hormone acting at distant sites.

Animals↗

PubMed1999

Requirements for heme and thiols for the nonenzymatic modification of nitrotyrosine.

Peroxynitrite-dependent formation of nitrotyrosine has been associated with inactivation of various enzymes and proteins possessing functionally important tyrosines. We have previously reported an enzymatic activity modifying the nitrotyrosine residues in nitrated proteins. Here we are describing a nonenzymatic reduction of nitrotyrosine to aminotyrosine, which depends on heme and thiols. Various heme-containing proteins can mediate the reaction, although the reaction also is catalyzed by heme. The reaction is most effective when vicinal thiols are used as reducing agents, although ascorbic acid also can replace thiols with lesser efficiency. The reaction could be inhibited by (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, but not other tested NO donors. HPLC with electrochemical detection analysis of the reaction identified aminotyrosine as the only reaction product. The reduction of nitrotyrosine was most effective at a pH close to physiological and was markedly decreased in acidic conditions. Various nitrophenol compounds also were modified in this reaction. Understanding the mechanism of this reaction could help define the enzymatic modification of nitrotyrosine-containing proteins. Furthermore, this also could assist in understanding the role of nitrotyrosine formation and reversal in the regulation of various proteins containing nitrotyrosine. It also could help define the role of nitric oxide and other reactive species in various disease states.

Chromatography, High Pressure Liquid↗

PubMed1999

Discovery of some of the biological effects of nitric oxide and its role in cell signaling.

The role of nitric oxide in cellular signaling in the past 22 years has become one of the most rapidly growing areas in biology with more than 20,000 publications to date. Nitric oxide is a gas and free radical with an unshared electron that can regulate an ever-growing list of biological processes. In many instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis from GTP. However, the list of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate its effect, while other reactions can lead to altered protein structure, function, and/or catalytic capacity. These diverse effects of nitric oxide that are either cyclic GMP dependent or independent can alter and regulate important physiological and biochemical events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an autacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique simple molecule with an array of signaling functions. However, as with any messenger molecule, there can be too little or too much of the substance and pathological events result. Some of the methods to regulate either nitric oxide formation, metabolism, or function have been in clinical use for more than a century as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870's. Current and future research with nitric oxide and cyclic GMP will undoubtedly expand the clinicians' therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide and cyclic GMP formation and metabolism. Such promise and expectations have obviously fueled the interests in these signaling molecules for a growing list of potential therapeutic applications.

Animals↗

PubMed1999

Nitrotyrosine formation with endotoxin-induced kidney injury detected by immunohistochemistry.

The presence of nitrotyrosine in the kidney has been associated with several pathological conditions. In the present study, we investigated nitrotyrosine formation in rat kidney after animals received endotoxin for 24 h. With lipopolysaccharide (LPS) treatment, immunohistochemical data demonstrated intense nitrotyrosine staining throughout the kidney. In spite of marked nitrotyrosine formation, the architectural appearance of tubules, glomeruli, and capillaries remained intact when examined by reticulin staining. Our data suggested that the marked staining of nitrotyrosine in proximal tubular epithelial cells was in the subapical compartment where the endocytic lysosomal apparatus is located. Thus a large portion of nitrotyrosine may come from the hydrolysis of nitrated proteins that are reabsorbed by the proximal tubule during the LPS treatment. We also found the colocalization of nitric oxide synthase (NOS-1) and nitrotyrosine within the macula densa of LPS-treated rats by using a double fluorescence staining method. In renal arterial vessels, vascular endothelial cells were more strongly stained for nitrotyrosine than vascular smooth muscle cells. Control animals without LPS treatment showed much less renal staining for nitrotyrosine. The general distribution of nitrotyrosine staining in control rat renal cortex is in the proximal and convoluted tubules, whereas the endothelial cells of vasa recta are major areas of nitrotyrosine staining in inner medulla. The renal distribution of nitrotyrosine in control and LPS-treated animals suggests that protein nitration may participate in renal regulation and injury in ways that are yet to be defined.

Animals↗

PubMed1999

Cellular signaling with nitric oxide and cyclic GMP.

During the past two decades, nitric oxide signaling has been one of the most rapidly growing areas in biology. This simple free radical gas can regulate an ever growing list of biological processes. In most instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis. However, the identification of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. The effects of nitric oxide can mediate important physiological regulatory events in cell regulation, cell-cell communication and signaling. Nitric oxide can function as an intracellular messenger, neurotransmitter and hormone. However, as with any messenger molecule, there can be too much or too little of the substance and pathological events ensue. Methods to regulate either nitric oxide formation, metabolism or function have been used therapeutically for more than a century as with nitroglycerin therapy. Current and future research should permit the development of an expanded therapeutic armamentarium for the physician to manage effectively a number of important disorders. These expectations have undoubtedly fueled the vast research interests in this simple molecule.

Animals↗