Mercury intoxication disrupts tonic signaling in B cells, and may promote autoimmunity due to abnormal phosphorylation of STIM-1 and other autoimmunity risk associated phosphoproteins involved in BCR signaling.

Epidemiological studies link exposure to mercury with autoimmune disease. Unfortunately, in spite of considerable effort, no generally accepted mechanistic understanding of how mercury functions with respect to the etiology of autoimmune disease is currently available. Nevertheless, autoimmune disease often arises because of defective B cell signaling. Because B cell signaling is dependent on phosphorylation cascades, in this report, we have focused on how mercury intoxication alters phosphorylation of B cell proteins in antigen-non stimulated (tonic) mouse (BALB/c) splenic B cells. Specifically, we utilized mass spectrometric techniques to conduct a comprehensive unbiased global analysis of the effect of inorganic mercury (Hg2+) on the entire B cell phosphoproteome. We found that the effects were pleotropic in the sense that large numbers of pathways were impacted. However, confirming our earlier work, we found that the B cell signaling pathway stood out from the rest, in that phosphoproteins which had sites which were affected by Hg2+, exhibited a much higher degree of connectivity, than components of other pathways. Further analysis showed that many of these BCR pathway proteins had been previously linked to autoimmune disease. Finally, dose response analysis of these BCR pathway proteins showed STIM1_S575, and NFAT2_S259 are the two most Hg2+ sensitive of these sites. Because STIM1_S575 controls the ability of STIM1 to regulate internal Ca2+, we speculate that STIM1 may be the initial point of disruption, where Hg2+ interferes with B cell signaling leading to systemic autoimmunity, with the molecular effects pleiotropically propagated throughout the cell by virtue of Ca2+ dysregulation.

Petroleum coke exposure leads to altered secretome profiles in human lung models.

Petroleum coke (PC) is a coal-like product that is produced during the refinement of crude oil and bituminous sand. Fugitive dust from open storage of PC in urban areas is a potential human health concern. Animal inhalation studies suggest that PC leads to an adverse pulmonary histopathology, including areas of fibrosis and chronic inflammation; however, little is known about its impact on human health. In order to identify biomarkers and cellular pathways that are associated with exposure, we performed two-dimensional liquid chromatography-mass spectrometric analyses on secreted proteins from two human lung culture models. A total of 2795 proteins were identified and relatively quantified from an immortalized cell line and 2406 proteins from primary cultures that were either mock treated or exposed to particulate matter with a diameter of 2.5-10 µm PC or filtered urban air particulates for 16 h. Pathway analysis on secretomes from primary lung cultures indicated that PC exposure suppressed the secretion of proteins involved in the organization of the extracellular matrix and epithelial differentiation. Because these cellular processes could facilitate fibrosis, we performed chronic 12-day exposure studies on three-dimensional human lung cultures consisting of epithelia and stromal fibroblasts. Relative to mock-treated cells, matrix metallopeptidase 9 levels in the conditioned media were lower by 4 days postexposure and remained suppressed for the duration of the experiment. Immunocytochemical staining of collagen III, a marker associated with fibrosis, showed increased accumulation in the epithelial layer and at the air-liquid interface.

Ca2+ binding site 2 in calcineurin-B modulates calmodulin-dependent calcineurin phosphatase activity.

Calcineurin is the Ca(2+)- and calmodulin-dependent Ser/Thr phosphatase. Human calcineurin-Aalpha and wild-type or mutated calcineurin-Bs were coexpressed in Escherichia coli and purified by calmodulin-Sepharose affinity chromatography. Four calcineurin-B mutants were studied. Each had a single conserved Glu in the 12th position of one EF-hand Ca(2+) binding site replaced by a Lys, resulting in the loss of Ca(2+) binding to that site. Phosphatase activities of the enzymes toward a (32)P-labeled phosphopeptide substrate were measured. Inactivating Ca(2+) binding sites 1, 2, or 3 in calcineurin-B reduced Ca(2+)-dependent phosphatase activity of the enzymes in the absence of calmodulin with the site 2 mutation being most effective. Inactivating Ca(2+) binding site 4 did not change enzyme activity or sensitivity to Ca(2+) in either the absence or presence of calmodulin. The calmodulin-dependent phosphatase activity of the enzymes containing site 1, 2, or 3 mutations in calcineurin-B was also decreased compared to enzyme with wild-type calcineurin-B. Of these enzymes, the one with the site 2 mutation was most profoundly affected as determined by the magnitude of the shift in Ca(2+) concentration dependence. Binding of a fluorescein-labeled calmodulin to the wild-type and the site 2 mutant enzymes was examined using fluorescence polarization measurements. The decrease in Ca(2+) sensitivity for the enzyme with calcineurin-B site 2 inactivated is apparently due to a decrease in the affinity of that enzyme for calmodulin at low Ca(2+) concentrations. These data support a role for Ca(2+) binding site 3 in the carboxyl half of calcineurin-B in transmitting the Ca(2+) signal to calcineurin-A and indicate that site 2 in the amino half of calcineurin-B is critical for enzyme activation.

Protein dephosphorylation rates in myocytes after isoproterenol withdrawal.

Dephosphorylation of substrates for cyclic AMP-dependent protein kinase is essential for reversing the effects of this enzyme. It has been proposed that the relevant phosphatase(s) is stimulated by muscarinic cholinergic agonists, thereby accentuating cholinergic antagonism of beta-adrenergic agonists in the heart. To test this hypothesis, dephosphorylation of the three major substrates of cardiac cyclic AMP-dependent protein kinase (phospholamban, troponin-I, and C-protein) was examined. In isolated myocytes, isoproterenol caused concentration-dependent phosphorylation of these three proteins. Simultaneous exposure to acetylcholine with the isoproterenol caused a rightward shift in the concentration-response curve that was similar for protein phosphorylation in myocytes and for the inotropic response of the intact heart. The addition of propranolol after exposure to isoproterenol resulted in protein dephosphorylation, the onset of which was accelerated by acetylcholine. However, acetylcholine did not affect the rate of dephosphorylation for any of the proteins, indicating that phosphatase activity in cardiac muscle is not enhanced by acetylcholine.

Modulation of the phosphatase activity of calcineurin by oxidants and antioxidants in vitro.

Previous research has indicated that oxidants, antioxidants and the intracellular redox state regulate the activities of a variety of protein tyrosine kinases, protein tyrosine phosphatases, phospholipases and transcription factors. In order to explore the redox regulation of the serine/threonine phosphatase calcineurin, we have investigated the effects of a variety of oxidants and antioxidants on calcineurin phosphatase activity in vitro. The oxidants hydrogen peroxide, superoxide and glutathione disulfide inhibited the phosphatase activity of calcineurin in a dose-dependent manner. Incubation of purified calcineurin with the antioxidants ascorbate, ascorbate 2-phosphate, alpha-lipoic acid, N-acetyl-L-cysteine and glutathione increased phosphatase activity relative to untreated controls. In contrast, several other commonly used antioxidants, including butylated hydroxytoluene, butylated hydroxyanisole, TEMPOL (4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl), Trolox (6-hydroxy-2,5,7, 8-tetramethyl-chroman-2-carboxylic acid) and dihydrolipoic acid decreased the activity of purified calcineurin, possibly through prooxidative mechanisms. Although the antioxidant pyrrolidine dithiocarbamate increased the activity of purified calcineurin, it significantly inhibited the activity of calcineurin present in crude fibroblast lysates. These results support and extend the hypothesis that redox factors modulate the phosphatase activity of calcineurin and suggest that further in vivo studies are warranted.

Ca(2+) binding and energy coupling in the calmodulin-myosin light chain kinase complex.

We have previously shown that 3 Ca(2+) ions are released cooperatively and 1 independently from the complex between (Ca(2+))4-calmodulin and skeletal muscle myosin light chain kinase or a peptide containing its core calmodulin-binding sequence. We now have found that three Ca(2+)-binding sites also function cooperatively in equilibrium Ca(2+) binding to these complexes. Replacement of sites I and II in calmodulin by a copy of sites III and IV abolishes these cooperative effects. Energy coupling-dependent increases in Ca(2+)-binding affinity in the mutant and native calmodulin complexes with enzyme are considerably less than in the peptide complexes, although the complexes have similar affinities. Ca(2+) binding to three sites in the native calmodulin-enzyme complex is enhanced; the affinity of the remaining site is slightly reduced. In the mutant enzyme complex Ca(2+) binding to one pair of sites is enhanced; the other pair is unaffected. In this complex reversal of enzyme activation occurs when Ca(2+) dissociates from the pair of sites with enhanced affinity; more rapid dissociation from the other pair has no effect, although both pairs participate in activation. Ca(2+)-independent interactions with calmodulin clearly play a major role in the enzyme complex, and appear to weaken Ca(2+)-dependent interactions with the core calmodulin-binding sequence.

Interactions of calcineurin A, calcineurin B, and Ca2+.

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.

Pyrethroid insecticides as phosphatase inhibitors.

In this study we tested the hypothesis that pyrethroid insecticides inhibit calcineurin directly and that inhibition is unaffected by the immunophilin cofactors necessary for calcineurin inhibition by cyclosporin A and FK506. The type II pyrethroid insecticides cis-cypermethrin (c-Cyp), trans-cypermethrin, deltamethrin (Delt), and fenvalerate A alpha (Fen), as well as the type I pyrethroid insecticides cis- and trans-permethrin and S-bioallethrin, were unable to inhibit the phosphatase activity of purified calcineurin under conditions of maximal activation by Ca2+ and calmodulin. Furthermore, c-Cyp, Delt, and Fen did not affect the Ca2+ dependence of calcineurin at 0.1 microM of calmodulin, indicating that Ca2+ binding to calmodulin was not affected by these agents. c-Cyp, Delt, and Fen also failed to inhibit calcineurin phosphatase activity in rat brain supernatant and cultured IMR-32 cells, although potent inhibition was displayed by both cyclosporin A and FK506 in each of these systems. Neither the Ca2+-dependent nor the okadaic acid-inhibitable phosphatase activity toward a 24-amino acid 32P-phospho-peptide substrate was affected by any of the pyrethroid insecticides, indicating that neither type-1 or type-2A phosphatase nor calcineurin is inhibited by pyrethroids. To determine if these results were dependent upon experimental conditions, experiments were repeated using polyethylene glycol-treated glass tubes in place of the standard polypropylene tubes. Regardless of the type of tube, no inhibition of calcineurin by any of the pyrethroid insecticides was observed. These data indicate that the pyrethroid insecticides are not effective inhibitors of calcineurin or other phosphatases.

Cyclosporin A has low potency as a calcineurin inhibitor in cells expressing high levels of P-glycoprotein.

Cyclosporin A (CsA) is a widely-used immunosuppressant drug whose therapeutic and toxic actions are mediated through inhibition of calcineurin (CN), a calcium- and calmodulin-dependent phosphatase. Inhibition of CN by CsA requires drug binding to its protein cofactor in the inhibition, cyclophilin. Because cyclophilin is a high affinity target for CsA it is expected that this protein can act as a reservoir for the drug in the cell and may be able to inhibit cellular efflux of CsA. P-glycoprotein (P-gp) is known to increase the rate of CsA efflux from CsA loaded cells but it is not clear if the P-gp drug efflux pump can compete effectively with cyclophilin at therapeutically relevant concentrations of CsA. To test the hypothesis that increased expression of P-gp confers protection against CsA-dependent inhibition of CN phosphatase activity, KB-V cells expressing varying levels of P-gp were analyzed to determine the potency of CsA as a CN inhibitor. When intact cells were treated with CsA, a positive correlation was observed between P-gp expression and resistance to CsA-dependent inhibition of CN: the IC50 is approximately 20-fold higher in the multidrug resistant epidermal carcinoma cell line, KB-V, which expresses P-gp at a high level than in the parental, KB, cell line expressing very low levels of P-gp. The resistance displayed by KB-V cells is abrogated by co-administration of the P-gp inhibitor verapamil, whereas verapamil has no effect on CsA potency in control KB cells. In cell lysates from KB-V cells with different amounts of P-gp CsA exhibits equivalent potency, indicating that the difference in sensitivity to CsA among the cell types requires maintenance of cell integrity. These observations support the view that resistance to CN inhibition by CsA occurs in cells with moderately elevated P-gp activity. Therefore, P-gp activity appears to be an important determinant of CsA cellular specificity for both therapeutic and toxic effects.

Localization of unique functional determinants in the calmodulin lobes to individual EF hands.

We have investigated the functional interchangeability of EF hands I and III or II and IV, which occupy structurally analogous positions in the native I-II and III-IV EF hand pairs of calmodulin. Our approach was to functionally characterize four engineered proteins, made by replacing in turn each EF hand in one pair by a duplicate of its structural analog in the other. In this way functional determinants we define as unique were localized to the component EF hands in each pair. Replacement of EF hand I by III reduces calmodulin-dependent activation of cerebellar nitric oxide synthase activity by 50%. Replacement of EF hand IV by II reduces by 60% activation of skeletal muscle myosin light chain kinase activity. There appear to be no major unique determinants for activation of these enzyme activities in the other EF hands. Replacement of EF hand III by I or IV by II reduces by 50-80% activation of smooth muscle myosin light chain kinase activity, and replacement of EF hand I by III or II by IV reduces by 90% activation of this enzyme activity. Thus, calmodulin-dependent activation of each of the enzyme activities examined, even the closely related kinases, is dependent upon a distinct pattern of unique determinants in the four EF hands of calmodulin. All the engineered proteins examined bind four Ca2+ ions with high affinity. Comparison of the Ca2+-binding properties of native and engineered CaMs indicates that the Ca2+-binding affinity of an engineered I-IV EF hand pair and a native I-II pair are similar, but an engineered III-II EF hand pair is intermediate in affinity to the native III-IV and I-II pairs, minimally suggesting that EF hands I and III contain unique determinants for the formation and function of EF hand pairs. The residues directly coordinating Ca2+ ion appear to play little or no role in establishing the different Ca2+-binding properties of the EF hand pairs in calmodulin.

Factors responsible for the Ca(2+)-dependent inactivation of calcineurin in brain.

The Ca(2+)-dependent protein phosphatase activity of crude rat brain extracts measured in the presence of okadaic acid, exhibits the characteristic properties of the calmodulin-stimulated protein phosphatase, calcineurin. It is stimulated more than 200-fold by Ca2+ and inhibited by the calmodulin-binding peptide, M13, and by the immunosuppressive drug, FK506. It is insensitive to rapamycin at concentrations up to 1 microM. Its specific activity, based on calcineurin concentration determined by quantitative analysis of Western blots exposed to anti-bovine brain IgG, is ten to twenty times that of purified rat brain calcineurin assayed under similar conditions. Unlike the purified enzyme it is rapidly and irreversibly inactivated in a time-, temperature-, and Ca2+/calmodulin-dependent fashion without evidence of extensive proteolytic degradation. The enzyme is converted to a state which does not lose activity by removal of low molecular weight material by gel filtration. Reconstitution of a labile enzyme is achieved by the addition of the low molecular weight-containing fraction eluted from the gel filtration column. These observations indicate that calcineurin in crude brain extracts is under the control of Ca2+/calmodulin-dependent positive and negative regulatory mechanisms which involve unidentified endogenous factor(s).

Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B.

The dependence of calcineurin on Ca2+ for activity is the result of the concerted action of calmodulin, which increases the turnover rate of the enzyme and modulates its response to Ca2+ transients, and of calcineurin B, which decreases the Km of the enzyme for its substrate. The calmodulin-stimulated protein phosphatase calcineurin is under the control of two functionally distinct, but structurally similar, Ca(2+)-regulated proteins, calmodulin and calcineurin B. The Ca(2+)-dependent activation of calcineurin by calmodulin is highly cooperative (Hill coefficient of 2.8-3), and the concentration of Ca2+ needed for half-maximum activation decreases from 1.3 to 0.6 microM when the concentration of calmodulin is increased from 0.03 to 20 microM. Conversely, the affinity of calmodulin for Ca2+ is increased by more than 2 orders of magnitude in the presence of a peptide corresponding to the calmodulin-binding domain of calcineurin A. Calmodulin increases the Vmax without changing the Km value of the enzyme. Unlike calmodulin, calcineurin B interacts with calcineurin A in the presence of EGTA, and Ca2+ binding to calcineurin B stimulates native calcineurin up to only 10% of the maximum activity achieved with calmodulin. The Ca(2+)-dependent activation of a proteolyzed derivative of calcineurin, calcineurin-45, which lacks the regulatory domain, was used to study the role of calcineurin B. Removal of the regulatory domain increases the Vmax of calcineurin, as does binding of calmodulin, but it also increases the affinity of calcineurin for Ca2+. Ca2+ binding to calcineurin B decreases the Km value of calcineurin without changing its Vmax.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoflurane isomers differ in preservation of ATP in anoxic rat hepatocytes.

The volatile anesthetic isoflurane inhibits the precipitous drop of ATP concentration that results from induced anoxia in isolated rat hepatocytes. This change is dose dependent and reaches its maximum at clinically relevant anesthetic concentrations. The use of highly purified isoflurane enantiomers has allowed a demonstration of the difference in the efficacy of the (+) and (-) isomers on the isoflurane-dependent preservation of ATP. The difference in relative potency between isomers was approximately 2.5-fold, with the (-) form being more potent. In a direct test of the anesthetic potency of these isomers, it was shown previously that the (+) isomer was more potent than the (-) isomer. Differences in the order of potency of isoflurane stereoisomers for the "anesthetic" and "metabolic" effects of isoflurane suggest that these effects are independent and may result from interactions with different specific molecular sites.

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.

Aging and digitalis sensitivity of cardiac muscle in rats.

The cause of the reduced tolerance of aged rats to the arrhythmogenic effect of ouabain was studied in left atrial and left ventricular muscle preparations obtained from 3-, 8- or 26-month old Fischer 344 rats. Under 1.5-Hz stimulation, the concentration-response curves for the positive inotropic effect of ouabain showed a tendency to shift to the left with age. The toxic effects occurred at lower ouabain or digoxin concentrations in the ventricular muscle obtained from senescent rats especially when the muscle was stimulated at a higher frequency (4 Hz), although differences in tolerance to the toxic effect of ouabain were not apparent in atrial muscle preparations stimulated at 1.5 Hz. No age-related difference in intracellular sodium or potassium concentration was observed in perfused ventricular muscle preparations paced at 1.5 Hz. There was no significant age effect on Na+-induced activation of Na+,K+-ATPase. In aged myocardium, however, the upstroke velocity of the action potential was reduced especially under high frequency stimulations, and the ability to follow high frequency stimulation was compromised. These results suggest that reserve capacity of the sodium pump is reduced in aged ventricular muscle, thereby elevating its sensitivity to the cardiac glycoside.

Influence of red blood cells, serum albumin, and serum lipoproteins on the clearance of benzo[alpha]pyrene by isolated livers of 3-methylcholanthrene-treated rats.

Red blood cells, serum albumin, and serum lipoproteins transport benzo[alpha]pyrene and other xenobiotic compounds in the circulation. The distribution of benzo[alpha]pyrene and its metabolites among these blood components was examined, and the effect of their presence in the perfusion medium on the ability of isolated livers from 3-methylcholanthrene-pretreated rats to clear circulating benzo[alpha]pyrene was determined. A large fraction (45%) of the benzo[alpha]pyrene in rat blood was associated with the serum lipoproteins. However, only 8% of the benzo[alpha]pyrene metabolites was associated with this component. Forty to forty-five percent of each was associated with red blood cells. Benzo[alpha]pyrene clearance by isolated rat livers was 1.8 +/- 0.2 ml/min when the medium contained only red blood cells and buffer. Addition of serum lipoproteins or serum albumin increased benzo[alpha]pyrene clearance to 5.1 +/- 0.5 or 8.5 +/- 0.9 ml/min respectively. Appearance of benzo[alpha]pyrene metabolites in perfusions medium and bile was similarly altered by the changes in medium composition. These results indicate that the clearance of benzo[alpha]pyrene by rat liver depends on the composition of the medium perfusing the organ and suggest that alterations in blood components in vivo may influence the metabolic disposition of this carcinogen.

Reduced tolerance to digitalis-induced arrhythmias caused by coronary-flow alterations in isolated perfused heart of guinea pigs.

The ligation of a main branch of coronary vein elevates the sensitivity of isolated, perfused (Langendorff) preparations of guinea-pig heart to arrhythmogenic actions of digoxin. Retrograde perfusion studies indicate that the presence of an area with a high extracellular K+ concentration, adjacent to an area with normal K+ concentration and exposed to the glycoside, predisposes the heart to digitalis-induced toxicity. This model may be useful in studying the mechanism by which acute myocardial infarction decreases the therapeutic index of cardiac glycosides.