Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase - PubMed (original) (raw)

Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase

N Ramachandran et al. Proc Natl Acad Sci U S A. 2001.

Abstract

N-dansylhomocysteine (DnsHCys) is quenched on S-nitrosation. The product of this reaction, N-dansyl-S-nitrosohomocysteine, is a sensitive, direct fluorogenic substrate for the denitrosation activity of protein disulfide isomerase (PDI) with an apparent K(M) of 2 microM. S-nitroso-BSA (BSA-NO) competitively inhibited this reaction with an apparent K(I) of 1 microM. The oxidized form of DnsHCys, N,N-didansylhomocystine, rapidly accumulated in cells and was reduced to DnsHCys. The fluorescence of DnsHCys-preloaded human umbilical endothelial cells and hamster lung fibroblasts were monitored as a function of extracellular BSA-NO concentration via dynamic fluorescence microscopy. The observed quenching of the DnsHCys fluorescence was an indirect measure of cell surface PDI (csPDI) catalyzed denitrosation of extracellular S-nitrosothiols as decrease or increase in the csPDI levels in HT1080 fibrosarcoma cells correlated with the rate of quenching and the PDI inhibitors, 5,5'-dithio-bis-3-nitrobenzoate and 4-(N-(S-glutathionylacetyl) amino)phenylarsenoxide inhibited quenching. The apparent K(M) values for denitrosation of BSA-NO by csPDI ranged from 12 microM to 30 microM. Depletion of membrane N(2)O(3) with the lipophylic antioxidant, vitamin E, inhibited csPDI-mediated quenching rates of DnsHCys fluorescence by approximately 70%. The K(M) for BSA-NO increased by approximately 3-fold and V(max) decreased by approximately 4-fold. These findings suggest that csPDI catalyzed NO released from extracellular S-nitrosothiols accumulates in the membrane where it reacts with O2 to produce N(2)O(3). Intracellular thiols may then be nitrosated by N2O3 at the membrane-cytosol interface.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Demonstration of PDI catalyzed DnsHCysNO fluorescence increase as a function of time. The fluorescence of 25 μM DnsHCysNO before (yellow circles) and after the addition of 0.25 μM PDI (green circles). The rate of fluorescence increase on addition of PDI, 0.34 ± 0.02/sec, was significant in comparison to the blank rate, 0.02 ± 0.01/sec (n = 4).

Figure 2

Figure 2

Plots of the initial rates of fluorescence increase, as a function of DnsHCysNO concentration in the presence of 0.25 μM PDI: no inhibitor (diamonds); or in the presence of 0.1 μM BSA-NO (squares), 1.0 μM BSA-NO (triangles), 10 μM BSA-NO (circles). Error bars represent SD (n = 6). The solid line represents the best fit of the data to the Michaelis–Menten equation.

Figure 3

Figure 3

DnsHCys fluorescence-quench kinetics. Fluorescence decrease was monitored as a function of time. (Insets) The semiln plots from which the _k_obs was estimated. The solid line represents the best fit of the data to a first-order process (n = 3). (A) DnsHCys2 (25 μM) was reduced with 100 μM GSH and treated with 200 μM NO(aq) (triangles) in a 3.0-ml stirred fluorescence cuvette (n = 3). (B) DnsHCys2 (25 μM) was reduced with 100 μM GSH and treated with 200 μM GSNO(aq) (circles), in a 3.0-ml stirred fluorescence cuvette (n = 3). (C) The fluorescence microscope images of fibroblast cells grown on coverslips, were acquired at 0.2-msec intervals subsequent to the introduction of 100 μM GSNO to the coverslip holder compartment. (D) The quenching rates for intracellular DnsHCys by 200 μM extracellular NO(aq) (squares) were extracted with the aid of

northern exposure

image processing software from the change in the intracellular fluorescence intensity/μm2 of microscope images of cells grown on cover slips collected at ≈250-msec intervals (n = 3). (E) The quenching rates for intracellular DnsHCys by 200 μM extracellular GSNO (diamonds) were extracted with the aid of

northern exposure

image processing software from the change in the intracellular fluorescence intensity/μm2 of microscope images of cells grown on cover slips collected at ≈250-msec intervals (n = 3).

Figure 4

Figure 4

Intracellular fluorescence intensity of DnsHCys2-treated HT1080 fibroblastoma cells on introduction of 100 μM BSA-NO (indicated by up arrow) to cells in which csPDI was underexpressed (triangles) or overexpressed (circles). Control cells transfected with vector alone are shown by the squares. Intracellular (fluorescence/μm2) was calculated from digitized images of the observation field taken every 250 msec with the aid of

northern eclipse

5.0 imaging software.

Figure 5

Figure 5

Kinetics of csPDI-catalyzed intracellular DnsHCys S-nitrosation. (A) Initial rates of intracellular DnsHCys fluorescence quenching as a function of BSA-NO concentration. Control HT1080 cells are indicated by circles, and HT1080 cells overexpressing csPDI are indicated by squares. The error bars represent SD (n = 6). The solid line represents the best fit of the data to the Michaelis–Menten equation. (B) Initial rates of intracellular DnsHCys fluorescence quenching as a function of BSA-NO concentration. HUVECs, no inhibitor (circles); 100 μM DTNB (squares). The error bars represent SD (n = 6). The solid line represents the best fit of the data to the Michaelis–Menten equation. (C) Initial rates of intracellular DnsHCys fluorescence quenching as a function of BSA-NO concentration. Hamster lung fibroblasts, no inhibitor (circles); 100 μM DTNB (squares). The error bars represent SD (n = 6). The solid line represents the best fit of the data to the Michaelis–Menten equation. (D) Dynamic fluorescence quenching of intracellular DnsHCys fluorescence on the addition of 200 μM BSA-NO (indicated by arrow). HUVECs, no inhibitor (triangles); 100 μM GSAO (circles); 100 μM 4-(_N_-(_S_-glutathionylacetyl)amino)benzoic acid (diamonds). The error bars represent SD (n = 6).

Figure 6

Figure 6

The effect of α-tocopherol on csPDI-catalyzed intracellular S-nitrosation. (A) The intracellular fluorescence of DnsHCys2-pretreated HUVECs, controls (circles), or HUVECs grown in the presence of 10 μM α-tocopherol for 14 h (squares), monitored subsequent to the extracellular addition of 25 μM BSA-NO (indicated by down arrow). (B) Initial rates of intracellular DnsHCys fluorescence quenching as a function of BSA-NO concentration. HUVECs, controls (circles), or HUVECs grown in the presence of 10 μM α-tocopherol for 14 h (squares). The error bars represent SD (n = 6). The solid line represents the best fit of the data to the Michaelis–Menten equation.

Figure 7

Figure 7

Postulated mechanism for intracellular S-nitrosation by csPDI-catalyzed NO released from extracellular RSNOs.

Similar articles

Cited by

References

    1. Wink D A, Cook J A, Kim S Y, Vodovotz Y, Pacelli R, Krishna M C, Russo A, Mitchell J B, Jourd'heuil D, Miles A M, Grisham M B. J Biol Chem. 1997;272:11147–11151. - PubMed
    1. Girard P, Potier P. FEBS Lett. 1993;320:7–8. - PubMed
    1. Stamler J S, Simon D I, Osborne J A, Mullins M E, Jaraki O, Michel T, Singel D J, Loscalzo J. Proc Natl Acad Sci USA. 1992;89:444–448. - PMC - PubMed
    1. Myers P R, Minor R L, Jr, Guerra R, Jr, Bates J N, Harrison D G. Nature (London) 1990;10, 345:161–163. - PubMed
    1. Park J K J, Kostka P. Anal Biochem. 1997;249:61–66. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources