Age-related declines in α-Klotho drive progenitor cell mitochondrial dysfunction and impaired muscle regeneration.

While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the "anti-aging" protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age.

Inhibition of prostaglandin synthesis after metabolism of menadione by cultured porcine endothelial cells.

We have examined the effects of menadione on porcine aortic endothelial cell prostaglandin synthesis. Addition of 1-20 microM menadione caused a dose- and time-dependent inhibition of stimulated prostaglandin synthesis with an IC50 of 5 microM at 15 min. Concentrations greater than 100 microM menadione were necessary to increase 51Cr release from prelabeled cells. Recovery of enzyme inactivated by menadione required a 6-h incubation in 1% serum. In a microsomal preparation, menadione was shown to have no direct effect on conversion of arachidonic acid to prostaglandins. In intact cells menadione caused only a 40% inhibition of the conversion of PGH2 to prostacyclin. Enzymes involved in the incorporation and the release of arachidonic acid were not affected by menadione (20 microM, 15 min). Menadione undergoes oxidation/reduction reactions in intact cells leading to partial reduction of oxygen-forming, reactive oxygen species. In our cells menadione was found to increase KCN-resistant oxygen consumption. Further, an increased accumulation of H2O2 was observed with a time course consistent with menadione-induced inhibition of prostaglandin synthesis. We conclude that menadione at sublethal doses caused inhibition of prostaglandin synthesis. The mechanism involves inactivation of PGH2 synthase by a reactive species resulting from metabolism of menadione by endothelial cells.

Novel triterpenoids suppress inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse macrophages.

We have synthesized more than 80 novel triterpenoids, all derivatives of oleanolic and ursolic acid, as potential anti-inflammatory and chemopreventive agents. These triterpenoids have been tested for their ability to suppress the de novo formation of two enzymes, inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2), using IFN-gamma-stimulated primary mouse macrophages or lipopolysaccharide (LPS)-activated RAW 264.7 macrophages as assay systems. Two synthetic oleananes, 3,12-dioxoolean-1-en-28-oic acid (TP-69) and 3,11-dioxoolean-1,12-dien-28-oic acid (TP-72), were highly active inhibitors of de novo formation of both iNOS and COX-2. Both TP-69 and TP-72 blocked the increase in iNOS or COX-2 mRNA induced by IFN-gamma or LPS. In addition, TP-72 suppressed NF-KB activation in primary macrophages treated with the combination of IFN-gamma and LPS or IFN-gamma and tumor necrosis factor. The 3-alpha(axial)-epimer of ursolic acid suppressed de novo formation of COX-2, in contrast to naturally occurring 3-beta(equatorial)-ursolic acid. Inhibitory effects of TP-69 or TP-72 on iNOS formation were not blocked by the glucocorticoid receptor antagonist RU-486, indicating that these triterpenoids do not act through the glucocorticoid receptor, nor does TP-72 act as an iNOS or COX-2 enzyme inhibitor when added to RAW cells in which synthesis of these two enzymes in response to LPS has already been induced. It may be possible to develop triterpenoids as useful agents for chemoprevention of cancer or other chronic diseases with an inflammatory component.

Recovery of porcine aortic endothelial cell prostaglandin synthesis following inhibition by sublethal concentrations of hydrogen peroxide.

Confluent monolayers of porcine aortic endothelial cells exposed for 10 min to 100 microM H2O2 lose their capacity to produce prostaglandins in response to addition of saturating exogenous arachidonic acid. Significant recovery of prostaglandin I2 and E2 synthesis occurred within 3 h and full enzymatic capacity returned by 6 h. Reducing the injury by exposure to half the amount of H2O2 allowed prostaglandin I2 production to recover to a greater extent in 3 h, while cells exposed for 60 min to either 0.5 or 1.0 mM H2O2 demonstrated no recovery. Pre-treatment with either actinomycin D or cycloheximide also prevented recovery following exposure to 100 microM peroxide. Injured cells did not recover when incubated with balanced salts after removal of peroxide, while incubation with medium 199 allowed for the complete return of synthetic capacity. Addition of 1% fetal calf serum in medium 199 did not facilitate recovery. Production of prostaglandins from endogenous arachidonic acid, released by either bradykinin or the ionophore A23187, was also inhibited by H2O2 exposure, however, full recovery of this stimulated synthesis occurred within 3 h. Cycloheximide pre-treatment completely inhibited recovery of bradykinin-induced prostaglandin I2 synthesis. These data demonstrate that sublethal concentrations of H2O2 irreversibly inactivate fatty acid cyclooxygenase and that synthesis of new enzyme is required for recovery. This return of activity occurs more rapidly for production of prostaglandins from endogenous arachidonic acid compared with production following addition of exogenous substrate.

Mechanism of bradykinin-stimulated prostacyclin synthesis in porcine aortic endothelial cells.

Porcine aortic endothelial cells studied at confluence were found to synthesize both prostacyclin and prostaglandin E2. Addition of arachidonic acid, bradykinin, the calcium ionophore A23187 or thrombin stimulated prostaglandin formation, whereas addition of angiotensin II did not. Bradykinin was found to stimulate very potently arachidonic acid release from cells prelabelled with [3H]arachidonate, the response being dose-dependent and half-maximal at 8 ng/ml. The rate of release of label (primarily arachidonate) from cells was increased by bradykinin (100 ng/ml) approximately 8-fold, with a return to control levels by 10 min. The calcium ionophore, A23187, similarly released [3H]arachidonic acid from prelabelled cells; the rate of release was approximately linear for 15 min. Both bradykinin and ionophore A23187 stimulated [3H]arachidonate release from endothelial cell phospholipids, an effect which was abolished in a dose-dependent manner by mepacrine. Release in response to bradykinin was prevented by incubation in Ca2+-free medium. Trifluoperazine, a compound which can inhibit calmodulin-mediated events, blocked the release of label stimulated by bradykinin. These data indicate that the likely mechanism of bradykinin-stimulated prostaglandin production in endothelial cells involves the activation of a phospholipase via a Ca2+-calmodulin-dependent pathway.

Inhibition of renin release by analogues of adenosine in rabbit renal cortical slices.

Renal cortical slices obtained from male New Zealand rabbits were used to investigate the role of adenosine in the regulation of renin release. Isoproterenol produced a significant (p less than 0.01), twofold to threefold increase in renin release, that was both dose-dependent and time-dependent. Addition of either the l-phenylisopropyl or the N6-ethylcarboxamido derivative of adenosine attenuated this stimulation at concentrations as low as 10(-9) M or 10(-8) M, respectively. Higher doses of d-phenylisopropyladenosine (10(-6) M) or adenosine (10(-5) M) were necessary to significantly reduce the beta-adrenergic response (p less than 0.01). Inhibition was absent in slices preincubated with 10(-5) M 8-phenyltheophylline, a concentration that had no effect on either basal or stimulated renin release. The site of inhibition appeared to be distal to beta-adrenergic and prostaglandin receptors since l-phenylisopropyladenosine (10(-8) M) blocked stimulation by selective beta-adrenergic receptor agonists, prenalterol (10(-6) M) or salbutamol (10(-5) M), and by prostaglandin E1. These data suggest that adenosine and its analogues inhibit renin release and that this inhibition may be mediated by a receptor-dependent action on a common point in the pathway leading to release.

Effects of losartan on blood pressure, plasma renin activity, and angiotensin II in volunteers.

Losartan is an orally active, nonpeptide angiotensin II (Ang II) (site-1) receptor antagonist. We conducted a multiple-dose study in healthy male volunteers to investigate the tolerability, blood pressure effects, and changes in plasma renin activity (PRA) and plasma Ang II concentration associated with once-daily administration of 100 mg losartan for a week. Subjects were studied on a standardized sodium diet (24-hour urinary sodium excretion, 98 +/- 37 [SD] mEq per 24 hours on the placebo run-in day). Measurements of blood pressure, heart rate, PRA, Ang II, and aldosterone were taken during a placebo run-in day and after single and multiple (7 days) daily doses of losartan (100 mg, n = 10) or placebo (n = 4). Ang II was measured specifically by high performance liquid chromatography coupled with radioimmunoassay. In subjects given losartan, respective decreases (systolic/diastolic) from run-in in supine blood pressure 6 hours after dosing were (mean +/- SD), compared with the placebo run-in day, first dose: -8.8 +/- 9.6/-6.8 +/- 5.0, last dose: -11.6 +/- 8.9/-7.0 +/- 4.8 mm Hg (p < 0.05 for all changes). At this 6-hour time point, corresponding increases from run-in in PRA were from 1.2 +/- 0.6 to 12.0 +/- 6.3 (first dose) and 9.6 +/- 4.9 (last dose) ng angiotensin I per milliliter per hour and in Ang II were from 4.3 +/- 1.7 to 72.4 +/- 33.3 and 45.7 +/- 14.1 pg/mL. All changes in PRA and Ang II were statistically significant within the losartan-treated group, and the biochemical changes were significantly greater than those in the placebo-treated group. The increment in Ang II was less after the last dose than after the first (p < 0.05). The drug was well tolerated by all subjects. These data indicate that, under the conditions of this study, losartan administration (100 mg/day for eight doses over 9 days) results in treatment-related decreases in blood pressure and increases in PRA and Ang II octapeptide.

Relationship between alpha 1-acid glycoprotein and lidocaine disposition in myocardial infarction.

The effects of myocardial infarction (MI) on lidocaine disposition were investigated in eight patients during a constant infusion of 2 mg/min. Plasma lidocaine binding and total plasma and free lidocaine concentrations were measured 12, 24, 36, and 48 hr after beginning therapy and were related to alpha 1-acid glycoprotein (AAG) concentrations. By 48 hr total plasma lidocaine and AAG concentrations had risen, as had plasma lidocaine binding. Because of enhanced binding, free lidocaine concentrations did not change significantly over this time. There was a correlation between AAG and the binding ratio for lidocaine (r = 0.87) and between AAG and total plasma lidocaine concentrations (r = 0.81). The data suggest that the rise in AAG seen after MI is responsible for enhanced plasma lidocaine binding and may, at least in part, be related to lidocaine cumulation.

Lidocaine plasma protein binding.

The percent of unbound lidocaine in the plasma of 24 healthy subjects was measured by equilibrium dialysis after addition of 3 microgram/ml C14 lidocaine hydrochloride. The percentage of unbound lidocaine varied from 19.9 to 38.8 (30.2 +/- 5, mean +/- SD) was inversely related to the concentration of alpha 1-acid glycoprotein (AAG) in the plasma (r = -0.931, p less than 0.001). The binding ratio (number of moles bound divided by number of moles unbound) of lidocaine was directly related to the plasma AAG concentration (r = 0.960, p less than 0.001). The binding ratio of lidocaine in solutions containing AAG but no albumin, prepared from the plasma of subjects in the study, was also directly related to the concentration of this acute-phase protein (r = 0.909, p less than 0.001). Human serum albumin solution (4 gm/100 ml) bound lidocaine to the extent of 20% under the same conditions. There was no relationship between the binding ratio of lidocaine and the albumin concentration in the plasma of the 24 subjects. In 7 normal subjects variation in AAG between 2 samples collected at least 1 mo apart was associated with a concomitant change in plasma lidocaine binding (r = 0.943, p less than 0.01). Thus even in normal subjects there is considerable interindividual and intraindividual variation in lidocaine binding, and measurements of AAG concentration in plasma may be a useful predictor of the extent of lidocaine plasma binding.

Acetylcholinesterase inhibition by zifrosilone: pharmacokinetics and pharmacodynamics.

OBJECTIVE: To determine the pharmacokinetics, pharmacodynamics and safety of the acetylcholinesterase inhibitor zifrosilone in healthy male volunteers. METHODS: Pharmacokinetics, pharmacodynamics, and tolerance of zifrosilone were studied in a double-blind, sequential, single-escalating-dose, randomized panel design. Each panel consisted of six subjects, with four subjects receiving zifrosilone (10, 30, 60, 90, 120, 150, 200, 250, and 300 mg orally) and two subjects receiving matching placebo. Serial blood samples were obtained for zifrosilone plasma concentrations and red blood cell acetylcholinesterase and butyrylcholinesterase activities. Participating subjects (n = 54) were men between the ages of 18 and 45 years. Each subject had a normal physical examination, electrocardiogram, serum chemistries, hematology, urinalysis, and test for human immunodeficiency virus at screening. RESULTS: A greater than proportionate increase in mean plasma concentration values for area under the curve from time zero to infinity was observed over the 200 to 300 mg dose range groups. Red blood cell acetylcholinesterase showed a dose-inhibition relationship, with a mean maximum inhibition of 20.9% at 10 mg that increased to 62.1% at 300 mg. Butyrylcholinesterase activity was relatively unaffected by zifrosilone (< 20% inhibition at 300 mg). For doses > or = 200 mg, an Emax pharmacodynamic model was used to describe the relationship between zifrosilone plasma concentration and red blood cell acetylcholinesterase inhibition (Emax = 83.8%; EC50 = 0.65 ng/ml). CONCLUSIONS: Zifrosilone showed dose-dependent pharmacokinetics after oral administration and was effective in causing selective inhibition of red blood cell acetylcholinesterase.

Oxidant-sensitive protein phosphorylation in endothelial cells.

Reactive oxygen is an important regulator of vascular cell biology; however, the mechanisms involved in transducing signals from oxidants in endothelial cells are poorly defined. Because protein phosphorylation is a major mechanism for signal transduction, cultured aortic endothelial cells were exposed to nonlethal concentrations of H2O2 to examine oxidant-sensitive changes in phosphorylation state. Addition of H2O2 increases the phosphorylation of the heat shock protein 27 (HSP27) within 2 min. This response is maximal by 20 min and remains constant for more than 45 min. Levels of intracellular free Ca2+ in endothelial cells did not change following addition of 100 microM H2O2, nor did the ability of the cells to respond to bradykinin. H2O2-induced phosphorylations were either not affected or were slightly increased in cells pretreated with PKC inhibitors (H-8, staurosporin, or calphostin c). Two-dimensional analysis of phosphoproteins from homogenates of 32P-labeled cells revealed that phorbol myristate acetate (PMA) did not cause the same degree of HSP27 phosphorylation as H2O2. Simultaneous addition of 10 eta M PMA and 50 microM H2O2 decreased the oxidant-stimulated phosphorylation of the most acidic HSP27 isoform. These data suggest that signal transduction for H2O2-sensitive endothelial cell responses are not only independent of PKC, but may also be suppressed by the action of the kinase.

Arsenic induces oxidant stress and NF-kappa B activation in cultured aortic endothelial cells.

Chronic exposure to low levels of environmentally derived arsenite are associated with vascular diseases, such as arteriosclerosis. However, the cellular and molecular mechanisms for vascular disease in response to arsenic are not known. These studies investigated the hypothesis that nonlethal levels of arsenic increase intracellular oxidant levels, promote nuclear translocation of trans-acting factors, and are mitogenic. Incubation of second passage vascular endothelial cells with less than 5 microM arsenite for 4 h increased incorporation of [3H]thymidine into genomic DNA, while higher concentrations failed to stimulate or inhibit DNA synthesis. Within 1 h following addition of noncytotoxic concentrations of arsenite, oxidants accumulated and thiol status increased. During this time period, there was increased nuclear retention of NF-kappa B binding proteins and nuclear translocation of NF-kappa B also occurred in response to 100 microM H2O2. Supershift analysis demonstrated that p65/p50 heterodimers accounted for the majority of proteins binding consensus kappa B sequences in cells treated with arsenite or oxidants. The antioxidants, N-acetylcysteine or dimethylfumaric acid, increased intracellular thiol status and prevented both oxidant formation and translocation of NF-kappa B binding proteins in response to arsenite. These data suggest that arsenite initiates vascular dysfunction by activating oxidant-sensitive endothelial cell signaling.

Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite.

Elevated levels of arsenite, the trivalent form of arsenic, in drinking water correlates with increased vascular disease and vessel remodeling. Previous studies from this laboratory demonstrated that environmentally relevant concentrations of arsenite caused oxidant-dependent increases in nuclear transcription factor levels in cultured porcine vascular endothelial cells. The current studies characterized the reactive species generated in these cells exposed to levels of arsenite that initiate cell signaling. These exposures did not deplete 5'-triphosphate, nor did they affect basal or bradykinin-stimulated intracellular free Ca2+ levels, indicating that they were not lethal. Electron paramagnetic resonance (EPR) spectroscopy, including spin trapping with carboxy-PTIO (cPTIO), demonstrated that 5 microM or less of arsenite did not increase *NO levels over a 30-min period relative to *NO release stimulated by bradykinin. However, these same levels of arsenite rapidly increased both oxygen consumption and superoxide formation, as measured by EPR oximetry and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), respectively. Pretreatment of the cells with DPI, apocynin, or superoxide dismutase abolished arsenite-stimulated DMPO-OH adduct formation. Finally arsenite increased extracellular accumulation of H2O2, measured as oxidation of homovanillic acid, with the same time and dose dependence, as seen for superoxide formation. These data suggest that superoxide and H2O2 are the predominant reactive species produced by endothelial cells after arsenite exposures that stimulate cell signaling and activate transcription factors.

Increased focal adhesion kinase- and urokinase-type plasminogen activator receptor-associated cell signaling in endothelial cells exposed to asbestos.

Exposure of low-passage endothelial cells in culture to nonlethal amounts of asbestos, but not refractory ceramic fiber-1, increases cell motility and gene expression. These changes may be initiated by the fibers mimicking matrix proteins as ligands for receptors on the cell surface. In the present study, 1- to 3-hr exposures of endothelial cells to 5 mg/cm2 of chrysotile asbestos caused marked cell elongation and motility. However, little morphological change was seen when chrysotile was added to cells pretreated with either mannosamine to prevent assembly of glycophosphatidylinositol (GPI)-anchored receptors or with herbimycin A to inhibit tyrosine kinase activity. Affinity purification of GPI-anchored urokinase-type plasminogen activator receptor (uPAR) from chrysotile-exposed cells demonstrated that asbestos altered the profile of proteins and phosphoproteins complexed with this receptor. Tyrosine kinase activities in the complexes were also increased by asbestos. Immunoprecipitations with selective monoclonal antibodies demonstrated that both chrysotile and crocidolite asbestos increase kinase activities associated with p60 Src or p120 focal adhesion kinase (FAK). Further, chrysotile also changed the profile of proteins and phosphoproteins associated with FAK in intact cells. These data suggest that asbestos initiates endothelial cell phenotypic change through interactions with uPAR-containing complexes and that this change is mediated through tyrosine kinase cascades.

Cell signaling pathways elicited by asbestos.

In recent years, it has become apparent that minerals can trigger alterations in gene expression by initiating signaling events upstream of gene transactivation. These cascades may be initiated at the cell surface after interaction of minerals with the plasma membrane either through receptorlike mechanisms or integrins. Alternatively, signaling pathways may be stimulated by active oxygen species generated both during phagocytosis of minerals and by redox reactions on the mineral surface. At least two signaling cascades linked to activation of transcription factors, i.e., DNA-binding proteins involved in modulating gene expression and DNA replication, are stimulated after exposure of lung cells to asbestos fibers in vitro. These include nuclear factor kappa B (NF kappa B) and the mitogen-activated protein kinase (MAPK) cascade important in regulation of the transcription factor, activator protein-1 (AP-1). Both NF kappa B and AP-1 bind to specific DNA sequences within the regulatory or promoter regions of genes that are critical to cell proliferation and inflammation. Unraveling the cell signaling cascades initiated by mineral dusts and pharmacologic inhibition of these events may be important for the control and treatment of mineral-associated occupational diseases.

Antiplatelet effects of MK-852, a platelet fibrinogen receptor antagonist, in healthy volunteers.

MK-852, a cyclic heptapeptide, is a potent platelet fibrinogen receptor antagonist. When administered to normal healthy male subjects by 1- and 4-hour constant rate intravenous infusions, it provides a generally well-tolerated and reversible means of inhibition of platelet function. At infusion rates of 1 microgram/kg/min for 1 hour and 0.44 microgram/kg/min for 4 hours, respectively, MK-852 extended baseline bleeding time by greater than 2.2-fold and 2.6-fold, inhibited ADP-induced platelet aggregation by 76% and 69%, and inhibited collagen-induced platelet aggregation by 65% and 67%, respectively. The pharmacokinetics of MK-852 include an elimination half-life of approximately 2 hours, total clearance of about 150 ml/min, and volume of distribution of about 18 liters. Examination of the relationship between MK-852 whole-blood concentration in vitro and inhibition of platelet aggregation showed an EC50 of about 55 ng/ml and a Hill coefficient of 1.55. The infusions were generally well tolerated, with no study drug-related changes in blood counts or biochemical profiles.

Identification and role of thiols in Toxoplasma gondii egress.

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase that is secreted into the parasitophorous vacuole where it appears to be essentially inactive in an oxidized form. Recent evidence shows that nucleoside triphosphate hydrolase can be activated by dithiothreitol in vivo. On reduction of the enzyme, there is a rapid depletion of host cell ATP. Previous results also demonstrate a dithiothreitol induced egress of parasites from the host cell with a concurrent Ca2+ flux, postulated to be a consequence of the release of ATP-dependent Ca2+ stores within the tubulovesicular network of the parasitophorous vacuole. Reduction of the nucleoside triphosphate hydrolase appears crucial for its activation; however, the exact mechanism of reduction/activation has not been determined. Using a variety of techniques, we show here that glutathione promoters activate a Ca2+ flux and decrease ATP levels in infected human fibroblasts. We further show the in vitro activation of nucleoside triphosphate hydrolase by endogenous reducing agents, one of which we postulate might be secreted into the PV by T. gondii. Our findings suggest that the reduction of the parasite nucleoside triphosphate hydrolase, and ultimately parasite egress, is under the control of the parasites themselves.

Gossypol effects on endothelial cells and tumor blood flow.

Isomers (-, +) of the antitumor agent gossypol (G) were studied for their ability to reduce tumor ATP and blood flow in rats bearing subcutaneously implanted pancreatic tumors. A 50% reduction in tumor ATP/Pi within ih of a single injection of -G was associated with a 60% decline in tumor blood flow. To determine if these changes in tumor physiology could be due to a direct drug effect on tumor endothelium, G isomers were compared for their ability to alter protein (125I-BSA) permeability and metabolic (32P) labelling of cultured endothelial cells. Treatments for ih produced no endothelial cell leakage, but 24h exposures to either -G (5 microM) or +G (50 microM) produced complete permeability of the monolayers to 125I-BSA. In contrast, 0.5-I.Oh exposures to -G (4 microM) produced 2 to 3-fold increases in phosphorylated 27 kDa heat-shock protein, hsp-27. Hsp-27 phosphoprotein isoforms were differentially labelled following -G and +G exposures with the phosphorylation profile of -G appearing most similar to that of oxyradical producing agents known to induce hsp-27 and injure endothelial cells. We postulate that the tumor ischemic effects of -G are mediated by endothelial response to oxyradical production in a mechanism similar to that of tissue ischemia-reperfusion injury.

A multiple-dose safety trial of eptastigmine in Alzheimer's disease, with pharmacodynamic observations of red blood cell cholinesterase.

A placebo-controlled multiple dose study was conducted to evaluate the safety, tolerability, and pharmacodynamics of multiple dose levels of eptastigmine in 25 patients with probable Alzheimer's disease (AD). Twenty patients (12 M, 8 F; mean age 74, range 57-84) were randomized to receive 12mg (N = 3), 20mg (N = 6), 28mg (N = 6) or placebo (N = 5) tid on a double-blind basis for 14 days, followed by seven days of single blind placebo, in successively rising dose groups. All patients completed the study without intolerable or severe adverse events. All doses significantly (p < 0.001) reduced peak and trough RBC cholinesterase (AChE) activity as compared to baseline. Percent inhibition for Day 14 peak and trough RBC AChE peak and trough values, respectively, appeared proportional to dose: 18% and 21% (12mg); 36% and 35% (20mg); 40% and 44% (28mg). In order to determine the maximum tolerated dose of eptastigmine, an additional single-blind study was performed in five patients (2 M, 3 F; mean age 78, range 72-80) utilizing a rising dose schedule of eptastigmine (N = 4) or placebo (N = 1), starting with the previously tolerated 28mg tid dose and increasing by 4mg tid up to a potential maximum of 56mg tid. Dose-limiting adverse events occurred requiring discontinuation of medication in one patient at 48mg tid and two patients at 52mg tid; RBC AChE inhibition was proportional to dose, with peak values up to 70% inhibition at 48mg tid. The maximum tolerated dose of 48mg tid was identified as a basis for potential Phase II multicenter efficacy trials.

Nickel-induced plasminogen activator inhibitor-1 expression inhibits the fibrinolytic activity of human airway epithelial cells.

One cause of debilitating pulmonary fibrosis is inhalation of insoluble metals. Human epidemiological and animal studies have associated inhalation of nickel dusts with increased incidence of pulmonary fibrosis. However, specific mechanisms for nickel-induced pulmonary fibrosis have yet to be elucidated. The current studies examine the hypothesis that particulate nickel promotes pulmonary fibrosis by inhibiting the fibrinolytic cascade. Since the urokinase-type plasminogen activator (uPA) initiates this cascade, this hypothesis was tested by investigating the effects of noncytotoxic levels of nickel subsulfide on the balance of uPA expression relative to expression of its inhibitor, PAI-1, in cultured human bronchial epithelial cells (BEAS-2B). Exposure to the metal decreased secreted uPA protein levels and activity without affecting uPA mRNA levels. In contrast, these same exposures stimulated transcription of PAI-1, causing prolonged increases in both mRNA and protein levels. Despite partial recovery of uPA protein levels, uPA activity remained depressed for more than 48 h after exposure to nickel due to the continued increase in PAI-1 expression. These data indicate that particulate nickel inhibits the fibrinolytic cascade by increasing the ratio of plasminogen inhibitor to activator. Sustained loss of uPA activity may contribute to nickel-induced pulmonary fibrosis in exposed populations.