Induced Reactive Oxygen Species Release Research Articles

Hydrogen peroxide diffusion and scavenging shapes mitochondrial network instability and failure by sensitizing ROS-induced ROS release

The mitochondrial network of cardiac cells is finely tuned for ATP delivery to sites of energy demand; however, emergent phenomena, such as mitochondrial transmembrane potential oscillations or propagating waves of depolarization have been observed under metabolic stress. While regenerative signaling by reactive oxygen species (ROS)-induced ROS release (RIRR) has been suggested as a potential trigger, it is unknown how it could lead to widespread responses. Here, we present a novel computational model of RIRR transmission that explains the mechanisms of this phenomenon. The results reveal that superoxide mediates neighbor-neighbor activation of energy-dissipating ion channels, while hydrogen peroxide distributes oxidative stress to sensitize the network to mitochondrial criticality. The findings demonstrate the feasibility of RIRR as a synchronizing factor across the dimensions of the adult heart cell and illustrate how a cascade of failures at the organellar level can scale to impact cell and organ level functions of the heart.

Effect of Indoxyl Sulfate on the Repair and Intactness of Intestinal Epithelial Cells: Role of Reactive Oxygen Species' Release.

Chronic kidney disease (CKD) is characterized by an oxidative stress status, driving some CKD-associated complications, even at the gastrointestinal level. Indoxyl Sulfate (IS) is a protein-bound uremic toxin, poorly eliminated by dialysis. This toxin is able to affect the intestinal system, but its molecular mechanism/s in intestinal epithelial cells (IECs) remain poorly understood. This study’s aim was to evaluate the effect of IS (31.2–250 µM) on oxidative stress in IEC-6 cells and on the intactness of IECs monolayers. Our results indicated that IS enhanced oxidative cell damage by inducing reactive oxygen species (ROS) release, reducing the antioxidant response and affecting Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation as well its related antioxidant enzymes. In the wound healing assay model, IS reduced IEC-6 migration, slightly impaired actin cytoskeleton rearrangement; this effect was associated with connexin 43 alteration. Moreover, we reported the effect of CKD patients’ sera in IEC-6 cells. Our results indicated that patient sera induced ROS release in IEC-6 cells directly related to IS sera content and this effect was reduced by AST-120 serum treatment. Results highlighted the effect of IS in inducing oxidative stress in IECs and in impairing the intactness of the IECs cell monolayer, thus significantly contributing to CKD-associated intestinal alterations.

Mitochondrial Membrane Potential Oscillations Persist during Reperfusion After Ischemia in MCU Knockout Cardiomyocytes

Ca2+ entry via Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload under physiological conditions but is thought to contribute to cell death during ischemia-reperfusion (I/R) injury. Previous work has shown that mitochondrial membrane potential (ΔΨm) instability contributes to early-reperfusion arrhythmias and contractile dysfunction; however, the role of mitochondrial Ca2+ uptake in triggering ΔΨm oscillation versus sustained permeability transition pore (PTP) opening is unclear. We hypothesized that MCU-mediated Ca2+ uptake is required to trigger irreversible ΔΨm loss mediated by PTP but is not required for ΔΨm oscillation during early reperfusion, involving reactive oxygen species (ROS)-induced ROS release (RIRR). A spinning-disk confocal microscope was used to image ΔΨm(with TMRM) in monolayers of neonatal mouse ventricular myocytes (NMVM) during I/R (1hr/1hr). Acute MCU knockout (KO) was achieved by transducing floxed MCU NMVMs with an adenovirus expressing CRE-recombinase. ΔΨm response to I/R was analyzed at cellular level by segmentation analysis (ImageJ) and a new wavelet transform method was developed to characterize the dominant frequency of ΔΨm oscillation (Matlab). We found that surprisingly, the rate of ΔΨm loss during ischemia was greater in MCU-KO compared to control, yet the recovery of ΔΨm was more sustained, lacking a late decline after 30 min reperfusion. ΔΨm oscillations during reperfusion persisted in MCU-KO myocytes and wavelet analysis revealed a dominant frequency ranging from 0.022Hz-0.0088Hz in both WT and KO. These results suggest that MCU-mediated Ca2+ uptake is not required for RIRR induced ΔΨm oscillation but is involved in late reperfusion-induced sustained ΔΨm collapse; and that MCU-mediated Ca2+ uptake during reperfusion contributes to PTP-mediated ΔΨm collapse during reperfusion. However, ΔΨm oscillations during early reperfusion are independent of MCU-mediated Ca2+ entry, and likely to underlie mechanical and electrophysiological dysfunction observed during this period.

The mitochondrial translocator protein and arrhythmogenesis in ischemic heart disease.

Mitochondrial dysfunction is a hallmark of multiple cardiovascular disorders, including ischemic heart disease. Although mitochondria are well recognized for their role in energy production and cell death, mechanisms by which they control excitation-contraction coupling, excitability, and arrhythmias are less clear. The translocator protein (TSPO) is an outer mitochondrial membrane protein that is expressed in multiple organ systems. The abundant expression of TSPO in macrophages has been leveraged to image the immune response of the heart to inflammatory processes. More recently, the recognition of TSPO as a regulator of energy-dissipating mitochondrial pathways has extended its utility from a diagnostic marker of inflammation to a therapeutic target influencing diverse pathophysiological processes. Here, we provide an overview of the emerging role of TSPO in ischemic heart disease. We highlight the importance of TSPO in the regenerative process of reactive oxygen species (ROS) induced ROS release through its effects on the inner membrane anion channel (IMAC) and the permeability transition pore (PTP). We discuss evidence implicating TSPO in arrhythmogenesis in the settings of acute ischemia-reperfusion injury and myocardial infarction.

Piperlongumine Blocks JAK2-STAT3 Pathway to Inhibit Collagen-Induced Platelet Reactivity Independent of Reactive Oxygen Species

Piperlongumine (PL) from piper longum induces cancerous, but not normal cells to undergo reactive oxygen species (ROS)-mediated apoptosis and blocks the activation of the transcription factor Stat3 in breast cancer cells. Paradoxically, this ROS-inducing agent also inhibits platelet activation and aggregation induced by collagen, which is reported to induce ROS production in platelets. Several types of ROS are known to activate platelets. We test the hypothesis that PL inhibits platelet reactivity to collagen by regulating a non-transcriptional activity of Stat3 that we have recently characterized. Consistent with previous reports, we found that PL dose-dependently blocked collagen-induced platelet activation, aggregation, and thrombus formation with a maximal inhibition at 100 mM. PL was equally effective in blocking platelet aggregation induced by a collagen-related peptide, suggesting that it primarily targets the collagen-receptor GP VI. Several lines of evidence suggest that this inhibitory effect of PL on collagen-induced platelet reactivity is mediated through blocking JAK2-Stat3 signaling. First, PL blocked the tyrosine phosphorylation of JAK2 and Stat3 in collagen-stimulated platelets. This inhibitory effect was significantly reduced in platelets that were pretreated with the Stat3 inhibitor STA21. Second, the JAK2 inhibitor AG490 blocked collagen-induced platelet aggregation and thrombus formation. Third, PL did not inhibit the tyrosine phosphorylation of Syk, the key kinase for collagen-GP VI signaling in platelets. However, it was effective in blocking PLCγ2 phosphorylation, a regulatory profile that is identical to that of Stat3 inhibitors to block collagen-induced platelet activation. Consistent with a report on cancer cells, PL at 100 mM induced platelets to release ROS detected by two specific probes and by a mass spectrometric measurement of platelet free glutathione (GSH). This effect was additive to that of collagen. Quenching oxidants by 20 µM of GSH (cell non-permeable) and 0.5 mM of L-Cysteine (cell permeable) did not block the inhibitory effects of PL. Neither was Apocynin, an inhibitor to reduced nicotinamide adenine dinucleotide phosphate, which blocks collagen-induced ROS production in platelets. The PL treated platelets were viable and responsive to other platelet agonists, suggesting that PL did not induce platelets to undergo apoptosis. Finally, two PL analogs that are active (MZ370) and inactive (MZ306) in inhibiting the growth of breast cancer cells were synthesized and found to be equally active in blocking collagen-induced platelet activation. In summary, we made several observations that define a mechanism for PL to inhibit collagen-induced platelet reactivity and provide new information regarding differential effects of ROS on platelet reactivity to collagen. First, we demonstrate that the inhibitory activity of PL on collagen-induced platelet reactivity was primarily mediated by blocking the activation of JAK2 and Stat3, but not a direct inhibition of collagen-GP IV signaling. Second, both PL and collagen induced ROS release in platelets, but PL’s inhibitory activity was not affected by ROS. The results suggest that PL and collagen induce the release of different oxidants that have differential effects on platelets. Studying these differential effects may uncover new mechanisms of regulating platelet functions by oxidants. DisclosuresNo relevant conflicts of interest to declare.

Biophysical properties and functional consequences of reactive oxygen species (ROS)‐induced ROS release in intact myocardium

Reactive oxygen species (ROS)-induced ROS release (RIRR) is a fundamental mechanism by which cardiac mitochondria respond to elevated ROS levels by stimulating endogenous ROS production in a regenerative, autocatalytic process that ultimately results in global oxidative stress (OS), cellular dysfunction and death. Despite elegant studies describing the phenomenon of RIRR under artificial conditions such as photo-induced oxidation of discrete regions within cardiomyocytes, the existence, biophysical properties and functional consequences of RIRR in intact myocardium remain unclear. Here, we used a semi-quantitative approach of optical superoxide (O(2)(-)) mapping using dihydroethidium (DHE) fluorescence to explore RIRR, its arrhythmic consequences and underlying mechanisms in intact myocardium. Initially, perfusion of rat hearts with 200 μM H(2)O(2) for 40 min (n = 4) elicited two distinct O(2)(-) peaks that were readily distinguished by their timing and amplitude. The first peak (P1), which was generated rapidly (within 5-8 min of H(2)O(2) perfusion) was associated with a relatively limited (10 ± 2%) rise in normalized O(2)(-) levels relative to baseline. In contrast, the second peak (P2) occurred 19-26 min following onset of H(2)O(2) perfusion and was associated with a significantly greater amplitude compared to P1. Spatio-temporal ROS mapping during P2 revealed active O(2)(-) propagation across the myocardium at a velocity of ~20 μm s(-1). Exposure of hearts (n = 18) to a short (10 min) episode of H(2)O(2) perfusion revealed consistent generation of P2 by high (≥200 μM, 8/8) but not lower (≤100 μM, 3/8) H(2)O(2) concentrations (P < 0.03). In these hearts, onset of P2 occurred following, not during, the 10 min OS protocol, consistent with RIRR. Importantly, P2 (+) hearts exhibited a markedly greater (by 3.8-fold, P < 0.001) arrhythmia score compared to P2 (-) hearts. To explore the mechanism underlying RIRR in intact myocardium, hearts were perfused with either cyclosporin A (CsA) or 4-chlorodiazepam (4-Cl-DZP) to inhibit the mitochondrial permeability transition pore (mPTP) or the inner membrane anion channel (IMAC), respectively. Surprisingly, perfusion with CsA failed to suppress (P = 0.75, n.s.) or even delay H(2)O(2)-induced P2 or the incidence of arrhythmias compared to untreated hearts. In sharp contrast, perfusion with 4-Cl-DZP markedly blunted O(2)(-) levels during P2, and suppressed the incidence of sustained ventricular tachycardia or ventricular fibrillation (VT/VF). Finally, perfusion of hearts with the synthetic superoxide dismutase/catalase mimetic EUK-134 completely abolished the H(2)O(2)-mediated RIRR response as well as the incidence of arrhythmias. These findings extend the concept of RIRR to the level of the intact heart, establish regenerative O(2)(-) production as the mediator of RIRR-related arrhythmias and reveal their strong dependence on IMAC and not the mPTP in this acute model of OS.

Vascular effects of Nox2‐derived radicals are dependent upon mitochondrial antioxidant capacity in old mice

Reactive oxygen species (ROS)-induced ROS release occurs when increased mitochondrial ROS generation increases NAD(P)H oxidase activity, and vice-versa. Whether this phenomenon contributes to age-related endothelial dysfunction is unknown. We tested the hypothesis that reductions in mitochondrial antioxidant capacity worsen endothelial function in old mice through augmented ROS-induced ROS release. Endothelial function and gene expression were examined in aorta from young (2 mo) and old (>18 mo) male MnSOD wild-type (WT) and MnSOD-deficient (HET) mice. Maximum relaxation to acetylcholine (MR, ACH) was unaffected in young HET mice (44 ± 3%) compared to WT mice (39% ± 3). Aging significantly impaired MR, ACH in WT mice (55 ± 3%), though MR, ACH did not differ between old WT and old HET mice (54% ± 3). Incubation with apocynin did not affect MR, ACH in young mice, but improved MR, ACH in old WT mice (43%± 3). Surprisingly, apocynin significantly impaired MR, ACH in old HET mice (60% ± 5). Expression of Nox2 was significantly increased in old mice, but unaffected by genotype. In conclusion, Nox2-derived ROS contribute to endothelial dysfunction in old mice with intact mitochondrial antioxidant defenses. Loss of mitochondrial antioxidant capacity with aging, however, appears to activate a Nox2-dependent, ROS-induced ROS-release cycle that paradoxically protects against endothelial dysfunction in old mice. NHLBI

Effects of single and multi walled carbon nanotubes on macrophages: Cyto and genotoxicity and electron microscopy

Production of nanotechnology-based materials is increasing worldwide: it is essential to evaluate their potential toxicity. Among these nanomaterials, carbon nanotubes (CNTs) have tremendous potential in many areas of research and applications. We have investigated the cyto- and genotoxic effects of single and multi-walled CNTs (SWCNTs, MWCNTs) and carbon black (CB) on the mouse macrophage cell line RAW 264.7. Specifically we have investigated inflammatory response, release of tumor necrosis factor-α (TNF-α), intracellular reactive oxygen species (ROS) production, cell death (both necrosis and apoptosis), chromosomal aberrations and cellular ultrastructural alteration caused by CB, MWCNTs and SWCNTs. Our data confirm that both CNTs and CB are cyto and geno-toxic to RAW 264.7 mouse macrophages. CNTs exposure induced ROS release, necrosis and chromosomal aberrations but did not cause an inflammatory response. In addition CNTs induce ultrastructural damage and apoptosis. CNTs penetrate the cell membrane and individual MWCNTs are seen associated with the nuclear envelope.

Cardiac arrhythmias induced by glutathione oxidation can be inhibited by preventing mitochondrial depolarization

We have previously proposed that the heterogeneous collapse of mitochondrial inner membrane potential (ΔΨm) during ischemia and reperfusion contributes to arrhythmogenesis through the formation of metabolic sinks in the myocardium, wherein clusters of myocytes with uncoupled mitochondria and high KATP current levels alter electrical propagation to promote reentry. Single myocyte studies have also shown that cell-wide ΔΨm depolarization, through a reactive oxygen species (ROS)-induced ROS release mechanism, can be triggered by global depletion of the antioxidant pool with diamide, a glutathione oxidant. Here we examine whether diamide causes mitochondrial depolarization and promotes arrhythmias in normoxic isolated perfused guinea pig hearts. We also investigate whether stabilization of ΔΨm with a ligand of the mitochondrial benzodiazepine receptor (4′-chlorodiazepam; 4-ClDzp) prevents the formation of metabolic sinks and, consequently, precludes arrhythmias. Oxidation of the GSH pool was initiated by treatment with 200 μM diamide for 35 min, followed by washout. This treatment increased GSSG and decreased both total GSH and the GSH/GSSG ratio. All hearts receiving diamide transitioned from sinus rhythm into ventricular tachycardia and/or ventricular fibrillation during the diamide exposure: arrhythmia scores were 5.5±0.5; n=6 hearts. These arrhythmias and impaired LV function were significantly inhibited by co-administration of 4-ClDzp (64 μM): arrhythmia scores with diamide+4-ClDzp were 0.4±0.2 (n=5; P<0.05 vs. diamide alone). Imaging ΔΨm in intact hearts revealed the heterogeneous collapse of ΔΨm beginning 20 min into diamide, paralleling the timeframe for the onset of arrhythmias. Loss of ΔΨm was prevented by 4-ClDzp treatment, as was the increase in myocardial GSSG. These findings show that oxidative stress induced by oxidation of GSH with diamide can cause electromechanical dysfunction under normoxic conditions. Analogous to ischemia–reperfusion injury, the dysfunction depends on the mitochondrial energy state. Targeting the mitochondrial benzodiazepine receptor can prevent electrical and mechanical dysfunction in both models of oxidative stress.

PU/PTFE-stimulated monocyte-derived soluble factors induced inflammatory activation in endothelial cells

Polyurethane (PU) and polytetrafluoroethylene (PTFE) are two commonly used blood-contacting biomaterials. In the present study, we used a noncontact coculture model to evaluate the thrombosis-causing potential of monocyte-mediated PU and PTFE. We used human endothelial cells from umbilical cord (HUVECs) and human monocytes (THP1 cells). The THP1 cells were directly exposed to PU/PTFE, and the resultant cell-free supernatants were harvested for stimulating HUVECs. The treated HUVECs constituted the test group. HUVECs treated with supernatants of LPS-stimulated THP1 cells were used as the positive controls. To investigate the effects of the supernatant treatment on HUVECs, we measured the expression of the leukocyte–endothelial-cell adhesion molecules (CAMs) CD54 (ICAM-1), CD106 (VCAM-1), and CD62E (E-selectin) and evaluated the release of tissue factor (TF). The results demonstrated that both PU and PTFE induced the expressions of CD62E and TF. These activation effects were accompanied by activation of the NF-κB transcription factor. To further investigate the monocyte-derived soluble factors that might contribute to these effects, we evaluated the effects of the PU/PTFE stimulation on the expression of reactive oxygen species (ROS), TNF-α, IL-1β, and IL-6 in monocyte monocultures. In comparison with the results for the negative control, both PU and PTFE significantly induced ROS release after 0.5 h, while the expressions of TNF-α, IL-1β, and IL-6 were variably increased after 24 h. Our results suggest that the biomaterial induces monocytic activation and subsequently causes the release of soluble factors, which contribute to the inflammatory activation in HUVECs.

A Wave of Reactive Oxygen Species (ROS)-Induced ROS Release in a Sea of Excitable Mitochondria

Once considered simply as the main source of ATP, mitochondria are now implicated in the control of many additional aspects of cell physiology, such as calcium signaling, and pathology, as in injury incurred on ischemia and subsequent reperfusion (I/R). Mitochondrial respiration is ordinarily accompanied by low-level ROS production, but they can respond to elevated ROS concentrations by increasing their own ROS production, a phenomenon termed ROS-induced ROS release (RIRR). Two modes of RIRR have been described. In the first mode of RIRR, enhanced ROS leads to mitochondrial depolarization via activation of the MPTP, yielding a short-lived burst of ROS originating from the mitochondrial electron transport chain (ETC). The second mode of RIRR is MPTP independent but is regulated by the mitochondrial benzodiazepine receptor (mBzR). Increased ROS in the mitochondrion triggers opening of the inner mitochondrial membrane anion channel (IMAC), resulting in a brief increase in ETC-derived ROS. Both modes of RIRR have been shown to transmit localized mitochondrial perturbations throughout the cardiac cell in the form of oscillations or waves but are kinetically distinct and may involve different ROS that serve as second messengers. In this review, we discuss the mechanisms of these different modes of RIRR.

Induction of mitochondrial ROS production by electrophilic lipids: a new pathway of redox signaling?

reactive oxygen species (ROS), such as superoxide or hydrogen peroxide, are usually thought of as damaging agents that nonspecifically disrupt proteins, lipids, and nucleic acids ([1][1]). However, there is now considerable evidence that ROS also act as signaling agents in a range of biological

218 Myocardial protection by ischaemic preconditioning and K(ATP) channels opening in the rat heart: evidence for involvement of tyrosine kinase and p38 MAPK

Background: Reactive oxygen species (ROS) contribute to the pathophysiology of chronic heart failure. A potential source of myocardial ROS may be the NAD(P)H oxidase. The small GTP-binding protein rac1 is a necessary regulator of the NAD(P)H oxidase. Thus, we examined ROS and rac1 in failing human myocardium and tested their regulation by statins in vivo. Methods and Results: Experiments were performed on human left ventricular myocardium from patients with ischemic (ICM) or dilated cardiomyopathy (DCM), compared to nonfailing controls (NF), n=8 per group. Furthermore, right atrial myocardium from patients undergoing cardiac surgery was used who were prospectively treated with atorvastatin or pravastatin (40 mg/day, respectively) or without statin, n=8 per group. Compared to NF, NAD(P)H oxidase activity was increased to 151 ± 28% in ICM and 153.9 ± 13% in DCM (p<0.05). Similarly, intracellular oxidative stress determined by aconitase activity assays was significantly increased in failing ventricles. This correlated (r=-0.69, p<0.001) with increased translocation of rac1 GTPase from the cytosol to the membrane in western-blot experiments (ICM, 142 ± 16%; DCM, 193 ± 19%, p<0.05 vs. NF). Pull-down assays showed a 3-fold increase of rac1 activity in ICM and DCM compared to NF myocardium (p<0.05). In parallel, membrane expression of the NAD(P)H oxidase subunit p47phox, but not p67phox, was upregulated in failing compared to nonfailing myocardium. In patients treated with statins, rac1 activity was decreased by 33 ± 12% (atorvastatin) and 35 ± 9% (pravastatin) compared to controls (p<0.05). Statin treatment did not alter basal NAD(P)H oxidase activity but significantly inhibited angiotensin II induced ROS release. Conclusions: In failing human myocardium, upregulation of NAD(P)H oxidase mediated ROS release is associated with increased rac1 membrane expression and activity, which can be reduced by oral statin treatment. These mechanisms may contribute to beneficial effects of statins in patients with chronic heart failure.

Involvement of oxygen radicals in cytarabine-induced apoptosis in human polymorphonuclear cells

We investigated apoptosis in polymorphonuclear neutrophils (PMNs) induced by cytarabine (Ara-C). This drug increased apoptosis by 100% with respect to the controls after 3 hr of incubation. This increase was inhibited by N-acetyl- l-cysteine (NAC) or diphenyleneiodonium chloride (DPI). Ara-C alone caused an early increase (after a 30-min incubation) in intracellular oxidant generation (inhibitable by rotenone, fumonisin b1, and DPI) and in protein tyrosine phosphorylations (inhibitable by NAC). The drug also affected the observed reduction of dimethylthiazol diphenyltetrazolium bromide (MTT). No extracellular release of reactive oxygen species (ROS) was elicited by the addition of Ara-C, while the drug increased the release of ROS by N-formyl-leucyl-phenylalanine-(f-MLP) but not phorbol 12-myristate 13-acetate-stimulated PMNs. This phenomenon was abolished by the addition of genistein, whereas such an effect was not observed following the addition of 1-(5-isoquinolynilsulfonyl)-2-methylpiperazine (H7). Ara-C induced ROS release from PMNs in the presence of subthreshold concentrations of f-MLP (priming effect). These results indicate that intracellular ROS production from mitochondria promotes Ara-C-induced apoptosis. Ara-C primes plasma membranes by a mechanism involving protein tyrosine phosphorylations and may also contribute to ROS generation from the granules.

Chronic Alcohol Intoxication Attenuates Human Immunodeficiency Virus‐1 Glycoprotein 120‐Induced Superoxide Anion Release by Isolated Kupffer Cells

This work tests the hypothesis that chronic alcohol intoxication suppresses the microbicidal activity of Kupffer cells by modulating the expression of cell surface receptors associated with respiratory burst and the release of potent microbicidal agents [i.e., reactive oxygen species (ROS)]. Because alcohol is also a potential risk factor in human immunodeficiency virus-1 (HIV-1) infection, this study examines the effect of HIV-1 glycoprotein 120 (gp120)-induced ROS release by isolated Kupffer cells. After 16 weeks of ethanol feeding, Kupffer cells from male Sprague-Dawley rats were isolated and assayed for HIV-1 gp120-induced superoxide release. Fluorescein isothiocyanate (FITC)-HIV-1 gp120 binding, NADPH oxidase, and protein kinase C activity in Kupffer cells were measured. Results show that HIV-1 gp120 induced the release of superoxide anion in a dose-dependent manner in normal rats. Mannosylated-bovine serum albumin inhibited FITC-HIV-1 gp120-mediated superoxide release in normal Kupffer cells by 85%. Moreover, 83 +/- 6% of Kupffer cells were FITC-HIV 1 gp120-positive, whereas <30% were CD4-positive. In alcohol-fed rats, HIV-1 gp120-induced ROS release was reduced by 70% and FITC-HIV-1 gp120 binding (in terms of fluorescence intensity per 10[6] Kupffer cells) by 44% in Kupffer cells, without any change in percent positive cells for this ligand. Concomitantly, HIV-1 gp120-induced translocation of NADPH oxidase to the plasma membranes of Kupffer cells in alcohol-fed rats was suppressed by 60%. In contrast, alcohol consumption significantly increased total protein kinase C activity and phorbol ester-induced superoxide release by Kupffer cells. These studies demonstrate that Kupffer cells are likely targets of HIV-1 whose binding sites on macrophages could also include mannose-specific receptors. These observations further suggest that suppression of HIV-1 gp120-mediated ROS production in chronic alcoholics is due to altered cell surface receptor expression for gp120, and defective postreceptor signaling mechanisms, which in turn could lead to attenuated microbicidal activity of hepatic macrophages.