496 Session 4: Nitric Oxide and Peroxynitrite

4:9 THE BIPHASIC EFFECT OF SODIUM NITROPRUSSIDE ON THE

OXIDATIVE MODIFICATION OF LDL BY MACROPHAGES.

B. Kalyanaraman , Ned Hogg , and Sampath Parthasarathy

Btophyslcs Research Institute, Medical College of Wisconsin,

Mdwaukae, WI 53226 and Dept. of Medicine, Umv. of Calif. San

!&go, Lalolla, CA 92093

Macrophage-medaated oxidation ot low-density hpoprotem (LDL)

has been suggested to be a key initial step m atherogenesis. Mechanistic

details of oxidative moditiclatlon of LDL by macrophages are not well

understood. Macrophages produce both superoxide (02q and nitric

oxide (.NO). the relatwe concentrations of which can be. Increased by

cytokmes. Recent reports suggest that cytokine-stimulated macrophages

oxldlzc LDL to a much lesser extent compared wth the unstlmulated

macrophages. This effect was attributed to an mcreased production of

.NO, whxh neutrahzed the eff&zt of 02T. To better understand the role

of mtnc oxide m macrophage-medlated oxldatmn of LDL. we have

mvestlgated the effect of .NO-relwmg agents such a\ >odlum

mtropruswlz (SNP) on LDL oxldatwn by macrophayrs. Whrn

macrophagzs were Incubated wth SNP, there was a dose-dependent

mcrcase m throbarbiturx aad rdactlve materials (TBARS) formed m the

madlum reaching a level of 1541 17% of the control levels. At concen-

trations below I mM, SNP (I) mcreased TBARS production

(II) mcreased the electrophoretlc mobibty of the mcubated LDL and (Iii)

enhanced Its degradation by macrophages. At higher concentrations of

SNP, there was actually an inhibition (> 70% at 5 m!vl and > 90% at

IO mM SNP) of the uptake of the mcubated LDL by macrophagas.

lnclwon of superoxide dismutase completely abolished the SNP-Induced

effects. Nitrlte concentrations were higher m mcubatmns contammg SNP

and macrophagas. We conclude that both 02’ and .NO are rqwnslblt:

for the blphasic effect of SNP on LDL oxldatmn.

4:ll INHIBITION OF SUPEROXIDE-DEPENDENT TOXICITY BY NITRIC OXIDE Bruce Freeman, Madia Trujillo, Homero Rubbo and Rafael Radi Dept. of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL 35233, USA and Dept. de Bioquimica, Univ. de la Republica, Montevideo, Uruguay.

Superoxide, nitric oxide and their reaction product peroxy- nitrite have all been shown to independently exert target tissue toxicity. Because these reactive species can be simultaneously generated in excess during different pathologic circumstances (acute inflammation, ischemia- reperfusion) we assessed the influence of nitric oxide on membrane lipid peroxidation induced by superoxide, hydrogen peroxide and hydroxyl radical derived from xanthine oxidase (X0) plus Fe-EDTA. Experimental conditions were adjusted to yield approximately 2 nmol/min superoxide and nitric oxide, with nitric oxide generated from the decomposition of S-nitroso-N-acetyl-penacillamine (SNAP). Peroxidation of liposomes made from a com- bination of egg and soybean phosphatidylcholine (1: 1, mol/mol) was assessed by formation of thiobarbituric acid positive products (TBA+). Nitric oxide alone did not induce lipid peroxidation, while exposure to hypoxanthine+XO+ Fe-EDTA produced up to 4 PM TBA+ materialfir. Liposomes exposed to simultaneous X0 and SNAP-derived reactive species had only control rates of lipid peroxidation, equivalent to that measured when DETAPAC was added to liposomes+HX+XO. We postulate that nitric oxide reacts with iron to inhibit subsequent prooxidative reactions of Fe+2 with hydrogen peroxide and alkyl hydroperoxides. It is concluded that the relative toxicities of superoxide, nitric oxide andperoxynitrite will deqendonlocal concentrations of oxidants and their mediators, as well as the indicator of target molecule toxicity which is being employed.

NITRONYL NITROXIDES TRAP NITRIC OXIDE AND

COUNTERACT THE VASORELAXANT EFFECTS OF NITRO-

VASODILATORS.

B. Kalyanaraman, Eugene A. Konorcv, Joy Joseph, and John E. Baker

Mrdxal College of W~sconsm, Milwaukcz, W~sconsm

Nltnc oxide (.NO) released from the mtrovasodilators, sodun

mtroprusslde (SNP) and S-nltroso-N-acetylpenicillamlna (SNAP), was

detected usmg an ,on,c mtronyl mtroxlde (SLI) and a non-nonlc mtronyl

mtroxlde (SLII). SNP 1s a negatively charged, divalent amomc molecule,

whereaa SNAP 1s a zwltter-Ion wrth a zero net charge. The purpose of

thla mvestlgatwn l> to tat thz follow& hypothws: I) nmonyl

mtroxlda, SLI and SLII, antagomzc the vasorelaxant effect of SNP and

SNAP, and II) this antagomsm 1s mlluenced differently by the mmc and

non-,on,c nature of SNP. SNAP, SLI and SLII.

SLL SL n

SLI and SLII trapped authentic .NO gas ,n solution and .NO released

tram SNP and SNAP formmg the correspondmg !mmo nitroxldes. This

reachon was followed by ESR spectral changes from a five-lme pattern

tor mtronyl mtroxldzs to a rune-lmc pattun tar ,m,nc nltroxide.\.

In wlated hearts, SNP (0.5-200 Fmol/l) and SNAP (2-20 +mol/l)

lncrzaaed coronary tlov. rate t,, a maximum of 185% and 190%,

raspcctwly. SNP-Induced vasoddatlon was InhIbItad by SLI (0.05-3.0

mmolil) from 162% to I3 I % of basrlme and by SLII from 185% to

146% of baseline values. However, SNAP-rnduced vasodllatlon was

antagomzed by SLII (from 190% to 136% of baselme at 20 pmolil

SNAP) but not by SLI (153 % vs 160% of baseline values). We conclude

that coronary vascular relaxation elntcd by SNP and SNAP IS mediated

by .NO and that the differential mhlbltory effwt of mtronyl mtroxides 1s

due to dlftercnces m locallzatwn ot nltronyl mtroxldea and nltrovaso-

dilators I” the coronary vasculature.

PEROXYNITRITE-MEDIATED CYTOTOXICITY

Homer0 Rubbo, Ana Denicola, Daniel Rodriguez, TO Trpanosoma cmi.

and Rafael Radi. Departamento de Bioquimica, Facultad de Medicina, Universidad de la Reptilica, Montevideo, URUGUAY.

Peroxynitrite anion, a reactive intermediate released by activated macrophages, was highly trypanocidal against epimastigote forms of T.cru.zi. Addition of micromolar steady-state concentrations of peroxynitrite resulted in inhibition of cell

9 roliferation as followed by

growth curves and [HI thymidine incorporation. Other cytotoxic effects of peroxynitrite included cellular swelling and inhibition of cell motility and respiration. We also observed that two critical enzymes for the energetic metabolism of the parasite, succinate dehydrogenase and NADH-fumarate reductase, were inactivated by biologically relevant concentrations of peroxynitrite. Direct reactions of peroxynitrite anion with critical sulfhydryl residues of the two enzymes were responsible for most of the observed inactivation as indicated by the protection afforded by peroxynitrite scavengers and the reactivation of the enzymes by dithiothreitol. We propose that peroxynitrite is a critical intermediate in the mechanisms of macrophage- mediated cytotoxicity to T.crui, exerting part of its cytotoxic effects through the inhibition of the energetic metabolism of the parasite.

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