Nitric oxide modulates endothelin 1-induced Ca2+ mobilization and cytoskeletal F-actin filaments in human cerebromicrovascular endothelial cells.
(17/20782)
A functional interrelation between nitric oxide (NO), the endothelial-derived vasodilating factor, and endothelin 1 (ET-1), the potent vasoconstrictive peptide, was investigated in microvascular endothelium of human brain. Nor-1 dose-dependently decreased the ET-1-stimulated mobilization of Ca2+. This response was mimicked with cGMP and abrogated by inhibitors of guanylyl cyclase or cGMP-dependent protein kinase G. These findings indicate that NO and ET-1 interactions involved in modulation of intracellular Ca2+ are mediated by cGMP/protein kinase G. In addition, Nor-1-mediated effects were associated with rearrangements of cytoskeleton F-actin filaments. The results suggest mechanisms by which NO-ET-1 interactions may contribute to regulation of microvascular function. (+info)
O-raffinose cross-linking markedly reduces systemic and renal vasoconstrictor effects of unmodified human hemoglobin.
(18/20782)
The hemodynamic effects of a 20% exchange-transfusion with different solutions of highly purified human hemoglobin A-zero (A0) were evaluated. We compared unmodified hemoglobin with hemoglobin cross-linked with O-raffinose. Unmodified hemoglobin increased systemic vascular resistance and mean arterial pressure more than the O-raffinose cross-linked hemoglobin solution (by approximately 45% and approximately 14%, respectively). Unmodified hemoglobin markedly reduced cardiac output (CO) by approximately 21%, whereas CO was unaffected by the O-raffinose cross-linked hemoglobin solution. Unmodified and O-raffinose cross-linked hemoglobin solutions increased mean arterial pressure to comparable extents ( approximately 14% and approximately 9%, respectively). Unmodified hemoglobin increased renal vascular resistance 2-fold and reduced the glomerular filtration rate by 58%. In marked contrast, the O-raffinose cross-linked hemoglobin had no deleterious effect on the glomerular filtration rate, renal blood flow, or renal vascular resistance. The extents to which unmodified and O-raffinose cross-linked hemoglobin solutions inactivated nitric oxide also were compared using three separate in vitro assays: platelet nitric oxide release, nitric oxide-stimulated platelet cGMP production, and endothelium-derived relaxing factor-mediated inhibition of platelet aggregation. Unmodified hemoglobin inactivated or oxidized nitric oxide to a greater extent than the O-raffinose cross-linked hemoglobin solutions in all three assays. In summary, O-raffinose cross-linking substantially reduced the systemic vasoconstriction and the decrease in CO induced by unmodified hemoglobin and eliminated the deleterious effects of unmodified hemoglobin on renal hemodynamics and function. We hypothesize that O-raffinose cross-linking reduces the degree of oxidation of nitric oxide and that this contributes to the reduced vasoactivity of this modified hemoglobin. (+info)
The central cannabinoid receptor (CB1) mediates inhibition of nitric oxide production by rat microglial cells.
(19/20782)
Upon activation, brain microglial cells release proinflammatory mediators, such as nitric oxide (NO), which may play an important role in the central nervous system antibacterial, antiviral, and antitumor activities. However, excessive release of NO has been postulated to elicit immune-mediated neurodegenerative inflammatory processes and to cause brain injury. In the present study, the effect of cannabinoids on the release of NO from endotoxin/cytokine-activated rat cortical microglial cells was evaluated. A drug dose-dependent (0.1 microM-8 microM) inhibition of NO release from rat microglial cells was exerted by the cannabinoid receptor high-affinity binding enantiomer (-)-CP55940. In contrast, a minimal inhibitory effect was exerted by the lower affinity binding paired enantiomer (+)-CP56667. Pretreatment of microglial cells with the Galphai/Galphao protein inactivator pertussis toxin, cyclic AMP reconstitution with the cell-permeable analog dibutyryl-cAMP, or treatment of cells with the Galphas activator cholera toxin, resulted in reversal of the (-)-CP55940-mediated inhibition of NO release. A similar reversal in (-)-CP55940-mediated inhibition of NO release was effected when microglial cells were pretreated with the central cannabinoid receptor (CB1) selective antagonist SR141716A. Mutagenic reverse transcription-polymerase chain reaction, Western immunoblot assay using a CB1 receptor amine terminal domain-specific antibody, and cellular colocalization of CB1 and the microglial marker Griffonia simplicifolia isolectin B4 confirmed the expression of the CB1 receptor in rat microglial cells. Collectively, these results indicate a functional linkage between the CB1 receptor and cannabinoid-mediated inhibition of NO production by rat microglial cells. (+info)
Effects of chronic nitric oxide activation or inhibition on early hepatic fibrosis in rats with bile duct ligation.
(20/20782)
Hepatic fibrosis or increased liver collagen contents drive functional abnormalities that, when extensive, may be life threatening. The purpose of this study was to assess the effects of the chronic stimulation or inhibition of nitric oxide synthesis in rats with hepatic fibrosis induced by permanent common bile duct ligation (3 weeks) and the role of expression of the different nitric oxide synthase isoforms. Bile duct ligation led to an important accumulation of collagen in the hepatic parenchyma, as shown both histologically and by the hydroxyproline contents of livers. Bilirubin and serum enzyme activities (measured as markers of cholestasis) increased several-fold after bile duct ligation. The area of fibrotic tissue, liver hydroxyproline content and serum markers of cholestasis were clearly related in obstructed rats. The absence of modifications in haemodynamic parameters excludes circulatory changes from being responsible for the development of liver alterations. In animals treated with NG-nitro-L-arginine methyl ester (L-NAME) the area of fibrosis was similar to that of untreated animals, the signs of cholestasis and cellular injury being more evident. In rats treated with L-arginine the area of fibrosis was almost three times larger than that found in bile duct ligated rats and in L-NAME-treated bile duct ligated rats, although the observed biochemical changes were similar to those seen in rats treated with L-NAME. Our results with inducible nitric oxide synthase, obtained by Western blots and immunohistochemistry, indicate a greater expression of the inducible enzyme in bile duct ligated and L-arginine-treated animals and a lower expression in the L-NAME and control groups. Constitutive nitric oxide synthase expression, obtained by Western blots, was very similar in all groups, except for the L-arginine-treated rats in which it was lower. These results suggest that nitric oxide production may be a key factor in the development of fibrosis in bile duct ligated rats. They also support the hypothesis of a dual role for nitric oxide; one beneficial, mediated by its circulatory effects, and the second negative, through its local toxic effects. (+info)
Nitric oxide inhibits cardiac energy production via inhibition of mitochondrial creatine kinase.
(21/20782)
Nitric oxide biosynthesis in cardiac muscle leads to a decreased oxygen consumption and lower ATP synthesis. It is suggested that this effect of nitric oxide is mainly due to the inhibition of the mitochondrial respiratory chain enzyme, cytochrome c oxidase. However, this work demonstrates that nitric oxide is able to inhibit soluble mitochondrial creatine kinase (CK), mitochondrial CK bound in purified mitochondria, CK in situ in skinned fibres as well as the functional activity of mitochondrial CK in situ in skinned fibres. Since mitochondrial isoenzyme is functionally coupled to oxidative phosphorylation, its inhibition also leads to decreased sensitivity of mitochondrial respiration to ADP and thus decreases ATP synthesis and oxygen consumption under physiological ADP concentrations. (+info)
L-arginine stimulation of glucose-induced insulin secretion through membrane depolarization and independent of nitric oxide.
(22/20782)
The mechanism of L-arginine stimulation of glucose-induced insulin secretion from mouse pancreatic islets was studied. At 16.7 mmol/l glucose, L-arginine (10 mmol/l) potentiated both phases 1 and 2 of glucose-induced insulin secretion. This potentiation of glucose-induced insulin secretion was mimicked by the membrane depolarizing agents tetraethylammonium (TEA, 20 mmol/l) and K+ (60 mmol/l), which at 16.7 mmol/l glucose obliterated L-arginine (10 mmol/l) modulation of insulin secretion. Thus L-arginine may potentiate glucose-induced insulin secretion by stimulation of membrane depolarization. At 3.3 mmol/l glucose, L-arginine (10 mmol/l) failed to stimulate insulin secretion. In accordance with membrane depolarization by the electrogenic transport of L-arginine, however, L-arginine (10 mmol/l) stimulation of insulin secretion was enabled by the K+ channel inhibitor TEA (20 mmol/l), which potentiates membrane depolarization by L-arginine. Furthermore, L-arginine (10 mmol/l) stimulation of insulin secretion was permitted by forskolin (10 micromol/l) or tetradecanoylphorbol 13-acetate (0.16 micromol/l), which, by activation of protein kinases A and C respectively sensitize the exocytotic machinery to L-arginine-induced Ca2+ influx. Thus glucose may sensitize L-arginine stimulation of insulin secretion by potentiation of membrane depolarization and by activation of protein kinase A or protein kinase C. Finally, L-arginine stimulation of glucose-induced insulin secretion was mimicked by NG-nitro-L-arginine methyl ester (10 mmol/l), which stimulates membrane depolarization but inhibits nitric oxide synthase, suggesting that L-arginine-derived nitric oxide neither inhibits nor stimulates insulin secretion. In conclusion, it is suggested that L-arginine potentiation of glucose-induced insulin secretion occurs independently of nitric oxide, but is mediated by membrane depolarization, which stimulates insulin secretion through protein kinase A- and C-sensitive mechanisms. (+info)
Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation.
(23/20782)
A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-i um-1, 2-diolate at concentrations from 10 to 1000 microM as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection. (+info)
Structural dynamics of ligand diffusion in the protein matrix: A study on a new myoglobin mutant Y(B10) Q(E7) R(E10).
(24/20782)
A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) --> Tyr, His(E7) --> Gln, and Thr(E10) --> Arg, called Mb-YQR], investigated by stopped-flow, laser photolysis, crystallography, and molecular dynamics (MD) simulations, proved to be quite unusual. Rebinding of photodissociated NO, O2, and CO from within the protein (in a "geminate" mode) allows us to reach general conclusions about dynamics and cavities in proteins. The 3D structure of oxy Mb-YQR shows that bound O2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O2 binding are reduced 10-fold. While there is no geminate recombination with O2 and CO, geminate rebinding of NO displays an unusually large and very slow component, which is pretty much abolished in the presence of xenon. These results and MD simulations suggest that the ligand migrates in the protein matrix to a major "secondary site," located beneath Tyr(B10)29 and accessible via the motion of Ile(G8)107; this site is different from the "primary site" identified by others who investigated the photolyzed state of wild-type Mb by crystallography. Our hypothesis may rationalize the O2 binding properties of Mb-YQR, and more generally to propose a mechanism of control of ligand binding and dissociation in hemeproteins based on the dynamics of side chains that may (or may not) allow access to and direct temporary sequestration of the dissociated ligand in a docking site within the protein. This interpretation suggests that very fast (picosecond) fluctuations of amino acid side chains may play a crucial role in controlling O2 delivery to tissue at a rate compatible with physiology. (+info)