Discovery of S-Nitrosoglutathione Reductase Inhibitors: Potential Agents for the Treatment of Asthma and Other Inflammatory Diseases

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A nonclinical safety and pharmacokinetic evaluation of N6022: a first-in-class S-nitrosoglutathione reductase inhibitor for the treatment of asthma

S-nitrosoglutathione reductase is the primary enzyme responsible for the metabolism of S-nitrosoglutathione (GSNO), the body's main source of bioavailable nitric oxide. Through its catabolic activity, GSNO reductase (GSNOR) plays a central role in regulating endogenous S-nitrosothiol levels and protein S-nitrosation-based signaling. By inhibiting GSNOR, we aim to increase pulmonary GSNO and induce bronchodilation while reducing inflammation in lung diseases such as asthma. To support the clinical development of N6022, a first-in-class GSNOR inhibitor, a 14-day toxicology study was conducted. Sprague-Dawley rats were given 2, 10 or 50 mg/kg/day N6022 via IV administration. N6022 was well tolerated at all doses and no biologically significant adverse findings were noted in the study up to 10 mg/kg/day. N6022-related study findings were limited to the high dose group. One male rat had mild hepatocellular necrosis with accompanying increases in ALT and AST and several male animals had histological lung assessments with a slight increase in foreign body granulomas. Systemic exposure was greater in males than females and saturation of plasma clearance was observed in both sexes in the high dose group. Liver was identified as the major organ of elimination. Mechanistic studies showed dose-dependent effects on the integrity of a rat hepatoma cell line.     

Effects of co‐exposure of lipopolysaccharide and β‐glucan (Zymosan A) in exacerbating murine allergic asthma associated with Asian sand dust

During the 2000s, Asian sand dust (ASD) was implicated in the increasing prevalence of respiratory disorders, including asthma. We previously demonstrated that a fungus from ASD aerosol exacerbated ovalbumin (OVA)‐induced airways inflammation. Exposure to heat‐inactivated ASD (H‐ASD) and either Zymosan A (ZymA, containing β‐glucan) or lipopolysaccharide (LPS) exacerbated allergic airways inflammation in a mouse model, but the effects of co‐exposure of LPS and β‐glucan are unclear. We investigated the effects of co‐exposure of LPS and ZymA in OVA‐induced allergic airways inflammation with ASD using BALB/c mice. Exposure to OVA + LPS enhanced the recruitment of inflammatory cells to the lungs, particularly neutrophils; exposure to OVA + LPS + H‐ASD potentiated this effect. Exposure to OVA + ZymA + H‐ASD stimulated the recruitment of inflammatory cells to the lungs, particularly eosinophils, and serum levels of OVA‐specific IgE and IgG1 antibodies, whereas exposure to OVA + ZymA did not affect most indicators of lung inflammation. Although exposure to OVA + LPS + ZymA + H‐ASD affected a few allergic parameters additively or synergistically, most allergic parameters in this group indicated the same level of exposure to OVA + LPS + H‐ASD or OVA + ZymA + H‐ASD. These results suggest that LPS and ZymA play different roles in allergic airways inflammation with ASD; LPS mainly enhances neutrophil recruitment through H‐ASD, and ZymA enhances eosinophil recruitment through H‐ASD.     

Formaldehyde dehydrogenase: Beyond phase I metabolism

Formaldehyde dehydrogenase, formally Class III alcohol dehydrogenase (ADH3), has recently been discovered to partially regulate nitrosothiol homeostasis by catalyzing the reduction of the endogenous nitrosylating agent S-nitrosoglutathione (GSNO). Several studies have implicated this enzyme, and in particular GSNO reduction, as playing an important role in conditions such as asthma, cardiovascular disease, and immune function. While ADH3 has received considerable attention in the biomedical literature where it is often referred to as GSNO reductase (GSNOR), ADH3-mediated GSNO reduction has received comparatively less attention in the environmental toxicology community. Herein, evidences for a role of ADH3 in cell signaling through thiol homeostasis is highlighted, underscoring that the enzyme functions more broadly than to metabolize formaldehyde.     

Spantide抑制甲醛炎性痛大鼠脊髓一氧化氮合酶表达和一氧化氮含量的增加(英文)

目的　观察鞘内注射P物质 (SP)拮抗剂spantide { [D Arg1,D Trp7,9,Leu11] substanceP}对炎性痛大鼠L5节段脊髓后角一氧化氮合酶 (NOS)表达和腰膨大一氧化氮 (NO)含量的影响 ,以探讨痛及痛过敏时脊髓NOS表达和NO生成增多的机制。方法　大鼠右后掌足底皮下注射 5 %甲醛 0 .2mL诱发炎性痛及痛过敏 ,NADPH d组化法观察脊髓后角NOS表达的变化 ,硝酸还原酶法测定NO含量的变化。结果　皮下注射甲醛 2 4h后 ,双侧L5节段脊髓后角NOS表达及腰膨大部位NO生成明显增加 ;注射甲醛前 5min鞘内注射spantide (5 μg ,10 μL) ,则明显抑制甲醛所致的NOS表达及NO生成增加。结论　初级传入末梢释放的SP在甲醛炎性痛及痛过敏时脊髓NOS表达及NO生成增多中发挥作用     

Erythrocyte nitric oxide synthase as a surrogate marker for mercury‐induced vascular damage: The modulatory effects of naringin

In this study, endothelial nitric oxide synthase activity and nitric oxide (NO) production by human erythrocytes in the presence and absence of mercuric chloride (HgCl₂), L ‐arginine (L ‐ARG), ‐ nitro‐L ‐arginine methyl ester (L ‐NAME), and naringin (NAR) were investigated. In addition, the levels of reduced glutathione (GSH) and related enzymes were estimated in erythrocytes hemolysate. The protein carbonyl content (PCC) and thiobarbituric acid‐reactive substances (TBARS) levels were also determined. The results of this study revealed that the treatment of erythrocytes with either HgCl₂ or L ‐NAME induced a significant decrease in NOS activity and nitrite levels compared with control cells. Furthermore, mercury exposure significantly increased the levels of PCC and TBARS but reduced the GSH level. The activities of glucose‐6‐phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, and glutathione‐S‐transferase (GST) were inhibited. The exposure of erythrocytes to HgCl₂ in combination with L ‐ARG, NAR, or both ameliorated the investigated parameters compared with erythrocytes incubated with HgCl₂ alone. These results indicate that mercury exposure decreased both erythrocyte NOS activity and nitrite production, and that these parameters might be indicative of mercury exposure. The data also suggest that concomitant treatment with NAR can restore NO bioavailability through either its metal‐chelating properties or its antioxidant activity. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1314–1322, 2014.     

齐墩果酸对氧化损伤模型人脐静脉内皮细胞的保护作用

目的:研究齐墩果酸对氧化损伤模型人脐静脉内皮细胞(HUVECs)的保护作用。方法:分别以含10%胎牛血清DMEM高糖培养液[含氧化低密度脂蛋白(ox-LDL,质量浓度分别为0、25、50、100、200、400μg/ml)]培养HUVECs,CCK-8法检测细胞活性以筛选复制模型最适质量浓度。以0、10、20、40、60、80、100μmol/L齐墩果酸培养HUVECs,CCK-8法检测细胞活性以考察齐墩果酸试验最适浓度。以5、10、20、40μmol/L齐墩果酸作用于氧化损伤模型HUVECs(100μg/ml ox-LDL诱导),CCK-8法检测细胞活性,测定一氧化氮(NO)、一氧化氮合酶(NOS)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)水平。结果:复制模型ox-LDL最适质量浓度为100μg/ml;齐墩果酸试验最适浓度范围为0⁴0μmol/L;5、10、20、40μmol/L齐墩果酸可增加氧化损伤模型HUVECs存活率,增强CAT、GSH-PX、NOS活性,增加NO含量。结论:齐墩果酸能够保护ox-LDL氧化损伤的HUVECs,其保护机制与增强CAT、GSH-PX、NOS抗氧化酶活性有关。     

Inhaled isobutyl nitrite inhibited macrophage inducible nitric oxide by blocking NFkappaB signaling and promoting degradation of inducible nitric oxide synthase-2

We previously reported that inhaled isobutyl nitrite inhibited macrophage tumoricidal activity by inhibiting inducible nitric oxide (NO) production. In the present study, a much shorter inhalant exposure regimen (five daily exposures) inhibited inducible NO and the NO synthase (NOS2). One of the ways in which NO and NOS2 are regulated is by ubiquitin-dependent NOS2 degradation. Immunoprecipitated NOS2 showed increased poly-ubiquitination, following exposure to the inhalant. In addition, Western blots of macrophage nuclear extracts for the NFkappaB subunit, p65, showed that exposure to the inhalant inhibited NFkappaB signaling, necessary for induction of NOS2. The inhalant blocked phosphorylation of the NFkappaB inhibitor, IkappaBalpha. The inhibition of NFkappaB signaling following inhalant exposure was confirmed using mice transgenic for the kappaB-dependent promoter of the HIV 5' LTR linked to luciferase. The data suggested that inhalant exposure likely inhibited macrophage NO production by blocking NFkappaB-mediated activation signaling and promoting poly ubiquitination of NOS2.     

The effects of thiol compounds and ebselen on nitric oxide activity in rat aortic vascular responses

1 Thiols have been implicated to play a role in a variety of aspects of nitric oxide (NO) generation and activity. Thiol dependence of nitric oxide synthase (NOS) has remained controversial and its mechanism is not clear. This study investigates possible mechanisms between thiol (SH group) and NOS activation, through thiol compounds (glutathione, dithiothreitol, N‐acetyl‐L ‐cysteine) and Ebselen [2‐phenyl‐1,2‐benzisoselenazole‐3(2H)‐one] on rat aortic vascular responses. 2 In rat thoracic aorta, acetylcholine (10^–6–10^–9 M) induced a relaxation of phenylephrine (PE) (10^–7 M )‐induced tone, which was inhibited dose dependently by increasing concentration of ebselen (1–10 μM ). 3 In rings of rat thoracic aorta, ebselen and NOS inhibitors (N^G‐monomethyl‐L ‐arginine, N^G‐nitro‐L ‐arginine methyl ester) produced an augmentation of phenylephrine (10^–7 M )‐ induced tone and acetylcholine induced a relaxation of PE (10^–7 M )‐induced tone in rat thoracic aorta, which was inhibited by ebselen (10 μM ) like NOS inhibitor. 4 The thiol compounds (glutathione, dithiothreitol, and N‐acetyl‐L ‐cysteine) alone did not change vascular tone in rat thoracic aorta. Pretreatment with thiol compounds before ebselen treatment, however, reversed the inhibitory effect of ebselen which acts like the NOS inhibitor in rat thoracic aorta. Posttreatment with thiol compounds after ebselen treatment did not reverse the inhibitory effect of ebselen by as much as pretreatment. 5 Calcium ionophore A23187 (10^–7 M )‐induced vasodilation was inhibited in ebselen pretreated rat thoracic aorta, but sodium nitroprusside (SNP, 10^–7 M )‐induced relaxation was not inhibited by ebselen. This suggests that NOS is involved in the inhibitory effect of ebselen on rat thoracic aorta relaxation. 6 These results suggest that ebselen exerts an inhibitory action on the nitric oxide synthesis in rat thoracic aorta by interacting with thiol groups.     

Acute effect of glucan-spiked office dust on nasal and pulmonary inflammation in guinea pigs

The acute effects of pure inhaled glucan on respiratory inflammation remain inconclusive and not sufficiently examined with regards to the simultaneous interaction of glucan, endotoxin (lipopolysaccharide, LPS), and house dust in airway inflammation. This study aims at determining effects of simultaneous exposure to office dust and glucan on nasal and pulmonary inflammation. This is relevant for humans with occupational exposure in waste handling and farming and buildings with mold problems. Office dust collected from Danish offices was spiked with 1% (1-3)-beta-glucan (curdlan). Guinea pig nasal cavity volume was measured by acoustic rhinometry (AR) and animals were exposed by inhalation for 4 h to curdlan-spiked dust, unspiked dust, purified air (negative controls), or LPS (positive controls). After exposure (+5 h) or the following day (+18 h), measurements were repeated by AR and followed by bronchoalveolar lavage (BAL). Total and differential cell counts, interleukin (IL)-8 in BAL fluid, and change in nasal volume were compared between groups. A 5-10% increase in nasal volume was seen for all groups including clean air except for a significant 5% decrease for spiked-dust inhalation (+18 h). No marked differences were observed in BAL cells or IL-8 except in LPS-exposed controls. The delayed decrease of nasal cavity volume after exposure to glucan spiked dust suggests a slow effect on the upper airways for curdlan and office dust together, though no pulmonary response or direct signs of inflammation were observed. Glucan-spiked office dust exposures produced a delayed nasal subacute congestion in guinea pigs compared to office dust alone, but extrapolated to nasal congestion in humans, paralleling the nasal congestion seen in human volunteers exposed to the same dust, this may not have clinical importance.     

PKC抑制剂灯盏花素乙对内脏炎症痛大鼠脊髓NO/cGMP信号转导系统的影响

目的探讨灯盏花素乙在甲醛复制的内脏炎症痛中的作用及其对NO/cGMP信号转导系统的影响。方法成年健康Wistar大鼠,随机分为4组:正常对照组、内脏炎症痛组、溶媒组和灯盏花素乙组。观察:①鞘内注射灯盏花素乙后大鼠行为学变化,以15min为一个时间段,共2h,计算疼痛分数;②致痛后30min取脊髓,用分光光度计法测定NOS活性、NO产量和放射免疫法测定cGMP含量。结果①灯盏花素乙组在前90min内疼痛分数低于内脏炎症痛组(P<0.05orP<0.01);②内脏炎症痛、溶媒和灯盏花素乙组脊髓的NOS活性、NO产量和cGMP含量均高于正常对照组(P<0.05orP<0.01),灯盏花素乙组低于内脏炎症痛组(P<0.01)。结论①灯盏花素乙能减轻甲醛复制的内脏炎症痛;②灯盏花素乙可能通过抑制脊髓内PKC的激活,进而抑制NO/cGMP信号转导系统的激活。     

Effects of isorhynchophylline on angiotensin II‐induced proliferation in rat vascular smooth muscle cells

Proliferation of vascular smooth muscle cells (VSMCs) is a crucial event in cardiovascular diseases. Isorhynchophylline, an alkaloid from a traditional Chinese medicine Gambirplant, has been used to treat cardiovascular diseases. The aim of this study was to investigate the effects of isorhynchophylline on angiotensin II (Ang II)‐induced proliferation of rat VSMCs. VSMCs were isolated from rat artery and cultured for 14 days before experimentation. The effect of isorhynchophylline on Ang II‐induced proliferation was evaluated by cell number, MTT assay and flow cytometry, and nitric oxide (NO) content and activity of NO synthase (NOS) were measured. The expression of proto‐oncogene c‐fos, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) mRNAs was measured by real‐time RT‐PCR. VSMC cultures were verified by morphology and immunostaining with α‐smooth muscle actin. Isorhynchophylline (0.1–10.0 μM) was not toxic to VSMCs, but markedly decreased Ang II (1.0 μm )‐enhanced cell number and MTT intensity, and blocked cell transition from G₀/G₁ to S phase. Furthermore, isorhynchophylline increased the NO content and NOS activity, and suppressed Ang II‐induced over‐expression of c‐fos, OPN and PCNA. Thus, isorhynchophylline was effective against Ang‐II induced cell proliferation, an effect that appears to be due, at least in part, to increased NO production, regulation of the cell cycle, and depressed expression of c‐fos, OPN and PCNA related to VMSC proliferation.     

Antidepressant activity and calcium signaling cascades

Although antidepressant treatments produce clear effects on monoaminergic neuronal function, the link between these effects and therapeutic response to treatment is controversial. Previous studies have demonstrated that antagonists of the NMDA receptor‐gated calcium ionophore result in antidepressant‐like responses in rodents and humans. Likewise, antidepressant treatments produce regionally selective adaptation of the NMDA receptor suggestive of diminished capacity to gate calcium into receptive neurons. Similarly, voltage‐dependent calcium channel antagonists have been reported to produce antidepressant‐like effects in rodents. A major target of increases in subcellular calcium concentration is nitric oxide synthase (NOS) which liberates NO in response to stimulation. Recently, we have demonstrated that nitric oxide synthase antagonists produced antidepressant‐like response in both in vivo preclinical screening procedures and in post‐mortem in vitro studies of β‐adrenoceptor density. We propose: 1) that interruption of the Ca²⁺‐calmodulin‐NOS‐guanylyl cyclase subcellular signaling pathway at any point will produce antidepressant‐like effects; 2) that the acute actions of antidepressants in preclinical screening procedures are a consequence of their ability to disrupt Ca²⁺‐calmodulin‐NOS‐guanylyl cyclase signaling; 3) that chronic but, not acute treatment with antidepressants results in adaptation of the Ca²⁺‐calmodulin‐NOS‐guanylyl cyclase signaling pathway; 4) that this adaptation is necessary for the achievement of the therapeutic actions of antidepressants and; 5) that major depression is accompanied by an alteration (hyperactivity?) of subcellular Ca²⁺ signaling. Copyright © 2001 John Wiley & Sons, Ltd.     

Acute toxicity and biochemical effects of azinphos methyl in the amphipod Hyalella curvispina

We evaluated the acute toxicity and biochemical effects of the organophosphorus pesticide azinphos methyl (AM) in the amphipod Hyalella curvispina that inhabits ponds and irrigation channels of an intensive fruit‐producing region in Rio Negro and Neuquén valley, North Patagonia, Argentina. The analysis by nonlinear regression of data from the 96 h‐acute toxicity tests indicated the coexistence of two subpopulations of H. curvispina with different susceptibilities to AM. The 96 h‐LC₅₀ for the resistant subpopulation (166 ± 56 μg/L) was 216‐fold higher than the 96h‐LC₅₀ value for the susceptible one (0.77 ± 1.33 μg/L).The two subpopulations could not be distinguished based on the biochemical measurements in control amphipods. Cholinesterase activity was significantly inhibited in AM‐exposed amphipods in a concentration‐dependent manner. The IC₅₀ value obtained after 96 h of exposure (2.18 ± 1.95 μg/L) was significantly lower than the 48 h‐IC₅₀ value (29.6 ± 17.4 μg/L). Carboxylesterase activity was significantly inhibited after 48 h of exposure to 12.5 and 62.5 μg/L AM (inhibition, 51%). This enzyme was thus able to protect cholinesterase from inhibition at 12.5 μg/L AM. Reduced glutathione and catalase showed a significant increase after 24 h of exposure as an adaptive response to AM, whereas glutathione S‐transferase activity was not significantly modified. The analysis of species sensitivity distribution showed that both subpopulations of H. curvispina were more tolerant to AM than most amphipod species, and that the susceptible subpopulation was more sensitive to AM than the other local aquatic species analyzed. The maximum concentration of AM in drainage water within the fruit‐producing area reported by other studies would affect most of the amphipod species (99%) and also a 44% of local aquatic ones. The results obtained in this study point out the usefulness of including amphipods like H. curvispina in ecotoxicity studies and monitoring programs to perform pesticide risk assessments. 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 1043–1053, 2014.     

Effect of exposure to volatile organic compounds (VOCs) on airway inflammatory response in mice

Volatile organic compounds (VOCs) are the main substances causing multiple chemical sensitivity reactions in human. The effects of single VOCs exposure on airway inflammatory responses in mice lung have been reported. Previous studies have demonstrated the role of reactive oxygen species (ROS) in lung inflammation induced by single VOCs inhalation. However, effects of VOCs exposure on NO signaling and neurological signaling pathways in airway remain less clear. We exposed male Kunming mice to filtered air (0) and four types of VOCs mixture (formaldehyde, benzene, toluene, and xylene) treated air. Group 1 is 1.0, 1.1, 2.0 and 2.0 mg/m³, group 2 is 3.0, 3.3, 6.0 and 6.0 mg/m³, group 3 is 5.0, 5.5, 10.0 and 10.0 mg/m³, group 4 is 10.0, 11.0, 20.0 and 20.0 mg/m³, which respectively corresponded to 10, 30, 50 and 100 times of indoor air quality standard in China 2 hr per day, 5 days per week, for 2 weeks in the whole body exposure chamber. One day following VOCs exposure, we collected lung, bronchoalveolar lavage fluid (BALF) from each mouse and examined oxidative stress markers, cellular infiltration and production of cytokines, neurotrophin and substance P. We found that VOCs exposure influenced significantly NOS activity, GSH, or IL-6 concentration. The number of total cells, macrophages and eosinophils increased significantly in group 4. In addition, the production of interferon-gamma (IFN-γ) and substance P were significantly decreased. In contrast, neurotrophin-3 production in BALF was significantly increased in group 3 and 4. Our findings suggest that NO signaling pathways may induce airway inflammatory in short term VOCs exposure mice and the airway inflammatory response may be modulated by neurological signaling.     

慢性神经痛大鼠鞘内联合应用吗啡和氯胺酮在脊髓上水平的抗伤害性作用及对吗啡耐受的影响

目的研究鞘内联合应用吗啡和氯胺酮对慢性神经痛大鼠的抗伤害作用机制及对吗啡耐受的影响。方法 40只Wistar大鼠,体质量220～260 g,制备坐骨神经结扎模型并进行鞘内置管,随机分为5组(n=8):B组为空白对照组;C组鞘内注射0.9%盐水10μL;K组鞘内注射氯胺酮50μg;M组鞘内注射吗啡20μg;KM组鞘内注射吗啡10μg和氯胺酮25μg。坐骨神经结扎术后第4天开始鞘内给药,每日1次,连续7 d。用药7d后处死大鼠,取大脑皮质、海马、脑干组织,硝酸还原酶法测定NO浓度和NOS活性。结果与B组比较,C和K组脑干NO浓度升高,M组皮质、海马、脑干中NO浓度均升高;与C组比较,M组皮质、海马NO浓度升高,KM组海马、脑干中NO浓度降低;与M组比较,KM组皮质、海马、脑干NO浓度均降低(P<0.05或0.01)。与B组比较,C、K和M组皮质、海马、脑干中NOS活性均升高;与C和M组比较,KM组皮质、海马、脑干中NOS活性均降低(P<0.05或0.01)。结论神经病理性痛大鼠鞘内联合应用吗啡和氯胺酮可在脊髓上水平产生抗伤害性作用,并可抑制吗啡耐受的形成,这可能与大脑皮质、海马、脑干的NO水平和NOS活性有关。     

Effect of perfluorooctanesulfonate exposure on steroid hormone levels and steroidogenic enzyme activities in juvenile Silurana tropicalis

The impact of the perfluoro‐chemical, perfluorooctanesulfonate (PFOS), on gonadal steroidogenesis during sexual differentiation in Silurana tropicalis was examined because of its ubiquity in the environment, bioaccumulative nature and potential to disturb endocrine activity. A partial life cycle study exposing S. tropicalis to varying concentrations of PFOS 0.06, 0.13, 0.25, 0.50 and 1.0 mg PFOS/L [nominal]) was conducted. Gonad and plasma samples were collected from juvenile control specimens and organisms exposed to PFOS from early embryo through 150 days post‐metamorphosis. Gonad CYP17, aromatase and 5α‐reductase activities were measured. Plasma estradiol, testosterone, dihydrotestosterone (DHT) and gonadal testosterone were measured in both males and females. Increased plasma DHT and gonadal testosterone were found in PFOS‐treated juvenile male S. tropicalis compared to controls. Decreased plasma estradiol, but not testosterone, was detected in PFOS‐treated female S. tropicalis compared to controls. Plasma DHT was not detected and an increase in gonadal testosterone was detected in PFOS‐treated female frogs. Female S. tropicalis exposed to PFOS exhibited a concentration‐related decrease in the mean aromatase activity, but not 5α‐reductase. PFOS exposure in male frogs induced a concentration‐related increase in 5α‐reductase activity, but did not alter aromatase activity compared to control frogs. A concentration‐related increase in CYP 17,20‐lyase activity, but not 17‐hydroxylase activity, was found in both female and male S. tropicalis exposed to PFOS.     

Effects of lead on Na+, K+‐ATPase and hemolymph ion concentrations in the freshwater mussel Elliptio complanata

Freshwater mussels are an imperiled fauna exposed to a variety of environmental toxicants such as lead (Pb) and studies are urgently needed to assess their health and condition to guide conservation efforts. A 28‐day laboratory toxicity test with Pb and adult Eastern elliptio mussels (Elliptio complanata) was conducted to determine uptake kinetics and to assess the toxicological effects of Pb exposure. Test mussels were collected from a relatively uncontaminated reference site and exposed to a water‐only control and five concentrations of Pb (as lead nitrate) ranging from 1 to 245 μg/L in a static renewal test with a water hardness of 42 mg/L. Endpoints included tissue Pb concentrations, hemolymph Pb and ion (Na⁺, K⁺, Cl^?, Ca²⁺) concentrations, and Na⁺, K⁺‐ATPase enzyme activity in gill tissue. Mussels accumulated Pb rapidly, with tissue concentrations increasing at an exposure‐dependent rate for the first 2 weeks, but with no significant increase from 2 to 4 weeks. Mussel tissue Pb concentrations ranged from 0.34 to 898 μg/g dry weight, were strongly related to Pb in test water at every time interval (7, 14, 21, and 28 days), and did not significantly increase after day 14. Hemolymph Pb concentration was variable, dependent on exposure concentration, and showed no appreciable change with time beyond day 7, except for mussels in the greatest exposure concentration (245 μg/L), which showed a significant reduction in Pb by 28 days, suggesting a threshold for Pb binding or elimination in hemolymph at concentrations near 1000 μg/g. The Na⁺, K⁺‐ATPase activity in the gill tissue of mussels was significantly reduced by Pb on day 28 and was highly correlated with tissue Pb concentration (R² = 0.92; P = 0.013). The Na⁺, K⁺‐ATPase activity was correlated with reduced hemolymph Na⁺ concentration at the greatest Pb exposure when enzyme activity was at 30% of controls. Hemolymph Ca²⁺ concentration increased significantly in mussels from the greatest Pb exposure and may be due to remobilization from the shell in an attempt to buffer the hemolymph against Pb uptake and toxicity. We conclude that Na⁺, K⁺‐ATPase activity in mussels was adversely affected by Pb exposure, however, because the effects on activity were variable at the lower test concentrations, additional research is warranted over this range of exposures. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.