A pilot study on the kinetics of metabolites and microvascular cutaneous effects of nitric oxide inhalation in healthy volunteers

RATIONALE: Inhaled nitric oxide (NO) exerts a variety of effects through metabolites and these play an important role in regulation of hemodynamics in the body. A detailed investigation into the generation of these metabolites has been overlooked. OBJECTIVES: We investigated the kinetics of nitrite and S-nitrosothiol-hemoglobin (SNO-Hb) in plasma derived from inhaled NO subjects and how this modifies the cutaneous microvascular response. FINDINGS: We enrolled 15 healthy volunteers. Plasma nitrite levels at baseline and during NO inhalation (15 minutes at 40 ppm) were 102 (86-118) and 114 (87-129) nM, respectively. The nitrite peak occurred at 5 minutes of discontinuing NO (131 (104-170) nM). Plasma nitrate levels were not significantly different during the study. SNO-Hb molar ratio levels at baseline and during NO inhalation were 4.7E-3 (2.5E-3-5.8E-3) and 7.8E-3 (4.1E-3-13.0E-3), respectively. Levels of SNO-Hb continued to climb up to the last study time point (30 min: 10.6E-3 (5.3E-3-15.5E-3)). The response to acetylcholine iontophoresis both before and during NO inhalation was inversely associated with the SNO-Hb level (r: -0.57, p = 0.03, and r: -0.54, p = 0.04, respectively). CONCLUSIONS: Both nitrite and SNO-Hb increase during NO inhalation. Nitrite increases first, followed by a more sustained increase in Hb-SNO. Nitrite and Hb-SNO could be a mobile reservoir of NO with potential implications on the systemic microvasculature

Identification of a new P1 residuemutation (444Arg----Ser) in a dysfunctional C1 inhibitor protein contained in atype II hereditary angioedema plasma

A new reactive-centre P1 residue mutation (444Arg----Ser), has been identified in a dysfunctional C1 inhibitor protein, C1 inhibitor(Ba), contained in a type II hereditary angioedema plasma. This substitution is compatible with a point mutation of the 444Arg codon (CGC----AGC), and represents the first non-histidine, non-cysteine P1 residue mutant described for C1 inhibitor

The catabolism of intact, reactive centre-cleaved and proteinase-complexed C1 inhibitor in the guinea pig

Clearance rates in the guinea pig were determined for intact guinea pig and human C1 inhibitor, the complexes of both inhibitors with human C1s, β factor XIIa and kallikrein, and for each inhibitor cleaved at its reactive centre with trypsin. Intact human and guinea pig C1 inhibitor were cleared from the circulation more slowly (t½s of 9.7 h and 12.1 h and fractional catabolic rates (FCRs) of 0.09 and 0.117) than any of their cleaved or complexed forms. The reactive centre-cleaved inhibitors were cleared with half-lives of 6.75 h for humans and 10.1 h for the guinea pig. The complexes with target proteases were catabolized much more rapidly, with half-lives ranging from 3.08 h to 4.3 h. The complexes with kallikrein were cleared more slowly than those with C1s and β factor XIIa. Complexes prepared with the guinea pig and human inhibitors were cleared at equivalent rates. The free inactivated proteases were cleared at rates similar to the equivalent complexes, except for kallikrein, which was cleared more rapidly than its complex. The fact that the complexes with different target proteases differed in their catabolism and that protease and complex catabolism were similar suggests that protease may play a direct role in clearance

Characterization of C1 inhibitor-Ta. A dysfunctional C1INH with deletion of lysine 251

Dysfunctional C1 inhibitor (C1INH)-Ta is a naturally occurring mutant from a patient with type II hereditary angioedema. This mutant has a deletion of the codon for Lys-251, which is located in the connecting strand between helix F and strand 3A, overlying beta sheet A. Deletion of this Lys modifies the amino acid sequence at this position from Asn-Lys-Ile-Ser to Asn-Ile-Ser and creates a new glycosylation site. To further characterize the mechanism of dysfunction, we have analyzed the recombinant normal and Ta proteins expressed by COS cells in addition to the proteins in serum and isolated from serum. Recombinant C1INH-Ta revealed an intermediate thermal stability in comparison with the intact and reactive center cleaved normal proteins. Analysis of the reactivity of this recombinant protein with target proteases demonstrated no complex formation with C1s, C1r, or kallikrein. Inefficient complex formation was, however, clearly detectable with beta-factor XIIa. Each protease produced partial cleavage of the recombinant mutant inhibitor. Recombinant C1INH-Ta, on 7.5% SDS-polyacrylamide gel electrophoresis and by size fractionation on Superose 12, showed a higher molecular weight fraction that was compatible in size with dimer formation. However, no multimerization of C1INH-Ta isolated from serum or of C1INH-Ta in serum, was observed. The C1INH-Ta dimer expressed the epitopes that normally are expressed only on the protease complexed or the cleaved inhibitor. These epitopes were not expressed on the monomeric inhibitor. The data suggest that the mutation in C1INH-Ta results in a folding abnormality that behaves as if it consists of two populations of molecules, one of which is susceptible to multimerization and one of which is converted to a substrate, but which retains residual inhibitory activit

Post-transcriptional regulation of the arginine transporter Cat-1 by amino acid availability

The regulation of the high affinity cationic amino acid transporter (Cat-1) by amino acid availability has been studied. In C6 glioma and NRK kidney cells, cat-1 mRNA levels increased 3.8-18-fold following 2 h of amino acid starvation. The transcription rate of the cat-1 gene remained unchanged during amino acid starvation, suggesting a post-transcriptional mechanism of regulation. This mechanism was investigated by expressing a cat-1 mRNA from a tetracycline-regulated promoter. The cat-1 mRNA contained 1.9 kilobase pairs (kb) of coding sequence, 4.5 kb of 3'-untranslated region, and 80 base pairs of 5'-untranslated region. The full-length (7.9 kb) mRNA increased 5-fold in amino acid-depleted cells. However, a 3.4-kb species that results from the usage of an alternative polyadenylation site was not induced, suggesting that the cat-1 mRNA was stabilized by cis-acting RNA sequences within the 3'-UTR. Transcription and protein synthesis were required for the increase in full-length cat-1 mRNA level. Because omission of amino acids from the cell culture medium leads to a substantial decrease in protein synthesis, the translation of the increased cat-1 mRNA was assessed in amino acid-depleted cells. Western blot analysis demonstrated that cat-1 mRNA and protein levels changed in parallel. The increase in protein level was significantly lower than the increase in mRNA level, supporting the conclusion that cat-1 mRNA is inefficiently translated when the supply of amino acids is limited, relative to amino acid-fed cells. Finally, y(+)-mediated transport of arginine in amino acid-fed and -starved cells paralleled Cat-1 protein levels. We conclude that the cat-1 gene is subject to adaptive regulation by amino acid availability. Amino acid depletion initiates molecular events that lead to increased cat-1 mRNA stability. This causes an increase in Cat-1 protein, and y(+) transport once amino acids become availabl

A hinge region mutation in C1-inhibitor (Ala436-->Thr) results in nonsubstrate-like behavior and in polymerization of the molecule

C1-inhibitor(Mo), a dysfunctional C1-inhibitor molecule produced in two kindred with type II hereditary angioedema, has a mutation at the P10 position (Ala436 to Thr). Like most serpins with hinge region mutations (P14, P12, P10), C1-inhibitor(Mo) loses its inhibitory activity. However, unlike the other hinge region mutations, this mutant is not converted to a substrate. As shown by nondenaturing gel electrophoresis, gel filtration, sucrose density gradient ultracentrifugation, and electron microscopy, C1-inhibitor(Mo) exists in both monomeric and multimeric forms. Polymerization probably results from reactive center loop insertion into the A sheet of an adjacent molecule. Native C1-inhibitor(Mo) was shown to have a thermal stability profile intermediate to those of intact and of cleaved normal C1-inhibitor. Native C1-inhibitor(Mo) did not bind to monoclonal antibody KII, which binds only to reactive center-cleaved normal C1-inhibitor. It did, however, react with monoclonal antibody KOK12, which recognizes complexed or cleaved C1-inhibitor but not intact normal C1-inhibitor. Native C1-inhibitor(Mo), therefore, exists in a conformation similar to the complexed form of normal C1-inhibito