Mechanism of inhibition of hepatic gluconeogenesis by bacterial endotoxin: a role for nitric oxide?

S-nitrosothiols are nitric oxide (NO)-derived molecules found in biological systems. They have been variously discussed as both NO reservoirs and as major actors in NO-dependent, but cGMP-independent, signal transduction. Although S-nitrosation of specific cysteine residues has been suggested to represent a novel redox-based signaling mechanism, the exact mechanisms of S-nitrosothiol formation under (patho)physiological conditions and the determinants of signaling specificity have not yet been established. Here we examined the sensitivity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to inhibition by S-nitrosocysteine (CysNO) and NO both intracellularly and in isolation. Bovine aortic endothelial cells (BAECs) and purified GAPDH preparations were treated with CysNO or NO, and enzymatic activity was monitored. Intracellular GAPDH was irreversibly inhibited upon CysNO administration, whereas treatment with NO resulted in a DTT-reversible inhibition of the enzyme. Purified GAPDH was inhibited by both CysNO and NO, but the inhibition pattern was diametrically opposite to that observed in the cells; CysNO-dependent inhibition was reversed with DTT, whereas NO-dependent inhibition was not. In the presence of GSH, NO inhibited purified GAPDH in a DTT-reversible way. Our data suggest that in response to CysNO treatment, cellular GAPDH undergoes S-nitrosation, which results in an irreversible inhibition of the enzyme under turnover conditions. In contrast, NO inhibits the enzyme via oxidative mechanisms that do not involve S-nitrosation and are reversible. In summary, our data show that GAPDH is a target for CysNO- and NO-dependent inhibition; however, these two agents inhibit the enzyme via different mechanisms both inside the cell and in isolation. Additionally, the differences observed between the cellular system and purified protein strongly imply that the intracellular environment dictates the mechanism of inhibition.

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On the Mechanism of Inhibition of CytochromecOxidase by Nitric Oxide

Journal of Biological Chemistry ◽

10.1074/jbc.271.52.33404 ◽

1996 ◽

Vol 271 (52) ◽

pp. 33404-33408 ◽

Cited By ~ 100

Author(s):

Alessandro Giuffrè ◽

Paolo Sarti ◽

Emilio D'Itri ◽

Gerhard Buse ◽

Tewfik Soulimane ◽

...

Keyword(s):

Nitric Oxide ◽

Mechanism Of Inhibition

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Unique regulation of anion/HCO3- exchangers by constitutive nitric oxide in rabbit small intestine

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00013.2003 ◽

2003 ◽

Vol 285 (6) ◽

pp. G1084-G1090 ◽

Cited By ~ 15

Author(s):

Steven Coon ◽

Uma Sundaram

Keyword(s):

Nitric Oxide ◽

Small Intestine ◽

Kinetic Studies ◽

Transport Processes ◽

Crypt Cell ◽

Maximal Rate ◽

Mechanism Of Inhibition ◽

Rabbit Small Intestine ◽

Villus Cell ◽

Inactive Analog

In the rabbit small intestine, there are three functionally different brush-border membrane (BBM) anion/HCO3- exchangers: 1) Cl/HCO3- exchange on the BBM of villus cells responsible for coupled NaCl absorption; 2) Cl/HCO3- exchange on the BBM of crypt cells possibly involved in HCO3- secretion; and 3) short-chain fatty acid (SCFA)/HCO3- exchange on the BBM of villus cells, which facilitates SCFA absorption. Although constitutive nitric oxide (cNO) has been postulated to alter many gastrointestinal tract functions, how cNO may specifically alter these three transporters is unknown. Inhibition of cNO synthase with NG-nitro-l-arginine methyl ester (l-NAME) 1) did not affect villus cell BBM Cl/HCO3 change, 2) stimulated crypt cell BBM Cl/HCO3- exchange, and 3) inhibited villus cell BBM SCFA/HCO3- exchange. d-NAME, an inactive analog of l-NAME, and l- N6-(1-iminoethyl)lysine, a more selective inhibitor of inducible NO, did not affect these transport processes. Kinetic studies demonstrated that 1) the mechanism of inhibition of crypt cell BBM Cl/HCO3- exchange is secondary to a decrease in the maximal rate of uptake of Cl, without an alteration in the affinity of the transporter for Cl, and 2) the mechanism of stimulation of villus cell BBM SCFA/HCO3- exchange is secondary to an increase in the affinity of the transporter for SCFA without an alteration in the maximal rate of uptake of SCFA. These results indicate that cNO uniquely regulates the three BBM anion/HCO3- transporters in the rabbit small intestine.

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The role of nitric oxide on decreasing the hepatic drug metabolizing enzyme activity by bacterial endotoxin in rats

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)34512-3 ◽

1999 ◽

Vol 79 ◽

pp. 125

Author(s):

Kiyoyuki Kitaichi ◽

Li Wang ◽

Haruna Kidokoro ◽

Mitsunori Iwase ◽

Kenji Takagi ◽

...

Keyword(s):

Nitric Oxide ◽

Enzyme Activity ◽

Bacterial Endotoxin ◽

Hepatic Drug ◽

Drug Metabolizing Enzyme ◽

Metabolizing Enzyme

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Na-glucose and Na-neutral amino acid cotransport are uniquely regulated by constitutive nitric oxide in rabbit small intestinal villus cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00124.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. G1030-G1035 ◽

Cited By ~ 15

Author(s):

Steven Coon ◽

James Kim ◽

Guohong Shao ◽

Uma Sundaram

Keyword(s):

Nitric Oxide ◽

Small Intestine ◽

Amino Acid ◽

Basolateral Membrane ◽

Kinetic Studies ◽

Mrna Levels ◽

Protein Levels ◽

Mechanism Of Inhibition ◽

Rabbit Small Intestine ◽

Villus Cell

Na-nutrient cotransport processes are not only important for the assimilation of essential nutrients but also for the absorption of Na in the mammalian small intestine. The effect of constitutive nitric oxide (cNO) on Na-glucose (SGLT-1) and Na-amino acid cotransport (NAcT) in the mammalian small intestine is unknown. Inhibition of cNO synthase with NG-nitro-l-arginine methyl ester (l-NAME) resulted in the inhibition of Na-stimulated 3H- O-methyl-d-glucose uptake in villus cells. However, Na-stimulated alanine uptake was not affected in these cells. The l-NAME-induced reduction in SGLT-1 in villus cells was not secondary to an alteration in basolateral membrane Na-K-ATPase activity, which provides the favorable Na gradient for this cotransport process. In fact, SGLT-1 was inhibited in villus cell brush-border membrane (BBM) vesicles prepared from animals treated with l-NAME. Kinetic studies demonstrated that the mechanism of inhibition of SGLT-1 was secondary to a decrease in the affinity for glucose without a change in the maximal rate of uptake of glucose. Northern blot studies demonstrated no change in the mRNA levels of SGLT-1. Western blot studies demonstrated no significant change in the immunoreactive protein levels of SGLT-1 in ileal villus cell BBM from l-NAME-treated rabbits. These studies indicate that inhibition of cNO production inhibits SGLT-1 but not NAcT in the rabbit small intestine. Therefore, whereas cNO promotes Na-glucose cotransport, it does not affect NAcT in the mammalian small intestine.

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Nitric oxide synthases: structure, function and inhibition

Biochemical Journal ◽

10.1042/bj3570593 ◽

2001 ◽

Vol 357 (3) ◽

pp. 593-615 ◽

Cited By ~ 1464

Author(s):

Wendy K. ALDERTON ◽

Chris E. COOPER ◽

Richard G. KNOWLES

Keyword(s):

Nitric Oxide ◽

Structure Function ◽

Protoporphyrin Ix ◽

Covalent Modification ◽

Exact Nature ◽

Mechanism Of Inhibition ◽

Wide Range ◽

Nos Inhibitors ◽

Other Information ◽

Made In

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, l-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

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Inhibition of the production of nitric oxide and vasodilator prostaglandins attenuates the cardiovascular response to bacterial endotoxin in adrenalectomized rats

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.1993.0108 ◽

1993 ◽

Vol 253 (1338) ◽

pp. 233-238 ◽

Cited By ~ 24

Keyword(s):

Nitric Oxide ◽

Cardiovascular Response ◽

Bacterial Endotoxin ◽

Adrenalectomized Rats

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Inducible Nitric Oxide Regulates Brush Border Membrane Na-Glucose Co-transport, but Not Na:H Exchange via p38 MAP Kinase in Intestinal Epithelial Cells

Cells ◽

10.3390/cells7080111 ◽

2018 ◽

Vol 7 (8) ◽

pp. 111 ◽

Cited By ~ 5

Author(s):

Palanikumar Manoharan ◽

Shanmuga Sundaram ◽

Soudamani Singh ◽

Uma Sundaram

Keyword(s):

Nitric Oxide ◽

Epithelial Cells ◽

Brush Border ◽

Brush Border Membrane ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Mechanism Of Inhibition ◽

Chronic Intestinal Inflammation ◽

Inducible Nitric Oxide

During chronic intestinal inflammation in rabbit intestinal villus cells brush border membrane (BBM) Na-glucose co-transport (SGLT1), but not Na/H exchange (NHE3) is inhibited. The mechanism of inhibition is secondary to a decrease in the number of BBM co-transporters. In the chronic enteritis mucosa, inducible nitric oxide (iNO) and superoxide production are known to be increased and together they produce abundant peroxynitrite (OONO), a potent oxidant. However, whether OONO mediates the SGLT1 and NHE3 changes in intestinal epithelial cells during chronic intestinal inflammation is unknown. Thus, we determined the effect of OONO on SGLT1 and NHE3 in small intestinal epithelial cell (IEC-18) monolayers grown on trans well plates. In cells treated with 100 μM SIN-1 (OONO donor) for 24 h, SGLT1 was inhibited while NHE3 activity was unaltered. SIN-1 treated cells produced 40 times more OONO fluorescence compared to control cells. Uric acid (1mM) a natural scavenger of OONO prevented the OONO mediated SGLT1 inhibition. Na+/K+-ATPase which maintains the favorable trans-cellular Na gradient for Na-dependent absorptive processes was decreased by OONO. Kinetics studies demonstrated that the mechanism of inhibition of SGLT1 by OONO was secondary to reduction in the number of co-transporters (Vmax) without an alteration in the affinity. Western blot analysis showed a significant decrease in SGLT1 protein expression. Further, p38 mitogen-activated protein (MAP) kinase pathway appeared to mediate the OONO inhibition of SGLT1. Finally, at the level of the co-transporter, 3-Nitrotyrosine formation appears to be the mechanism of inhibition of SGLT1. In conclusion, peroxynitrite inhibited BBM SGLT1, but not NHE3 in intestinal epithelial cells. These changes and the mechanism of SGLT1 inhibition by OONO in IEC-18 cells is identical to that seen in villus cells during chronic enteritis. Thus, these data indicate that peroxynitrite, known to be elevated in the mucosa, may mediate the inhibition of villus cell BBM SGLT1 in vivo in the chronically inflamed intestine.

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