Methylmercury interferes with glucocorticoid receptor: Potential role in the mediation of developmental neurotoxicity.

Methylmercury (MeHg) is a widespread environmental contaminant with established developmental neurotoxic effects. Computational models have identified glucocorticoid receptor (GR) signaling to be a key mediator behind the birth defects induced by Hg, but the mechanisms were not elucidated. Using molecular dynamics simulations, we found that MeHg can bind to the GR protein at Cys736 (located close to the ligand binding site) and distort the conformation of the ligand binging site. To assess the functional consequences of MeHg interaction with GR, we used a human cell line expressing a luciferase reporter system (HeLa AZ-GR). We found that 100 nM MeHg does not have any significant effect on GR activity alone, but the transactivation of gene expression by GR upon Dex (a synthetic GR agonist) administration was reduced in cells pre-treated with MeHg. Similar effects were found in transgenic zebrafish larvae expressing a GR reporter system (SR4G). Next we asked whether the effects of developmental exposure to MeHg are mediated by the effects on GR. Using a mutant zebrafish line carrying a loss-of-function mutation in the GR (grS357) we could show that the effects of developmental exposure to 2.5 nM MeHg are mitigated in absence of functional GR signaling. Taken together, our data indicate that inhibition of GR signaling may have a role in the developmental neurotoxic effects of MeHg.

Vanadate and phosphate ions reduce tension and increase cross-bridge kinetics in chemically skinned heart muscle.

Tension development, immediate stiffness and ATPase of chemically skinned myocardial strips were measured in solutions with varying concentrations of phosphate (Pi) or vanadate (predominantly H2VO4 at pH 7) ion. Vanadate and Pi decreased stiffness in proportion to tension. The results show that, like Pi, vanadate accelerates the turnover rate of cross-bridges, but is effective at about 1/500 the concentration required for the Pi effect. Both Pi and vanadate increased the energy cost of isometric tension maintenance (that is, the ratio of ATPase to tension) and increased the velocity of delayed tension development following quick stretch of the chemically skinned myocardial strips. The results also show that changes in the rate of rise of delayed tension during stretch activation probably reflect changes in the kinetics of the biochemical cycle of the cross-bridges.

Towards a molecular understanding of the elasticity of titin.

Vertebrate striated muscle behaves elastically when stretched and this property is thought to reside primarily within the giant filamentous protein, titin (connectin). The elastic portion of titin comprises two distinct structural motifs, immunoglobulin (Ig) domains and the PEVK titin, which is a novel motif family rich in proline, glutamate, valine and lysine residues. The respective contributions of the titin Ig and the PEVK sequences to the elastic properties of the molecule have been unknown so far. We have measured both the passive tension in single, isolated myofibrils from cardiac and skeletal muscle and the stretch-induced translational movement of I-band titin antibody epitopes following immunofluorescent labelling of sites adjacent to the PEVK and Ig domain regions. We found that with myofibril stretch, I-band titin does not extend homogeneously. The Ig domain region lengthened predominantly during small stretch, but such lengthening did not result in measurable passive tension and might be explained by straightening, rather than by unfolding, of the Ig repeats. At moderate to extreme stretch, the main extensible region was found to be the PEVK segment whose unravelling was correlated with a steady passive tension increase. In turn, PEVK domain transition from a linearly extended to a folded state appears to be principally responsible for the elasticity of muscle fibers. Thus, the length of the PEVK sequence may determine the tissue-specificity of muscle stiffness, whereas the expression of different Ig domain motif lengths may set the characteristic slack sarcomere length of a muscle type.

Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin-I mutants and troponin-C.

Troponin C (TnC) could be extracted from skinned porcine cardiac muscle fibres and their Ca2+ sensitivity restored by reconstitution with recombinant human cardiac TnC. After extraction of troponin I (TnI) and TnC using the vanadate treatment method of Strauss et al. [Strauss, J. D., Zeugner, C., Van Eyk, J.E., Bletz, C., Troschka, M. and Rüegg, J.C. (1992) FEBS Lett. 310, 229-234], skinned porcine cardiac muscle fibres were reconstituted with wild-type recombinant human cardiac TnC and either wild-type cardiac TnI or several mutant isoforms of human TnI. Reconstitution with wild-type proteins restored the Ca2+ sensitivity of the tissue and phosphorylation of the TnI with the catalytic subunit of protein kinase A reduced the Ca2+ sensitivity (i.e.-log[Ca2+] for 50% of maximal force) as has been shown by others. However, reconstitution with the TnI mutant Ser-23Asp/Ser-24Asp mimicking the phosphorylated form of cardiac TnI, led to a reduced Ca2+ sensitivity compared with reconstitution with wild-type TnI, whereas the mutant Ser-23Ala/Ser-24Ala behaved as the dephosphorylated form of TnI. These data confirm the importance of negative charge in this region of the TnI molecule in altering the Ca2+ responsiveness in this system.

Peptide competition of actin activation of myosin-subfragment 1 ATPase by an amino terminal actin fragment.

The amino-terminal region of actin participates in the binding of myosin subfragment 1 (S1) during cross-bridge cycling, thereby assisting in the activation of the magnesium-dependent myosin ATPase. Effects of three actin fragments on the magnesium-dependent S1 and acto-S1 ATPase activities in solution were studied. One of the peptides, containing residues actin 1-44, mimicked the S1 ATPase-activating properties of actin and in turn inhibited acto-S1 ATPase both in a concentration-dependent manner. This suggests peptide competition for the actin binding site on myosin. The other fragments, residues actin 1-18 and 82-119, respectively, had no detectable effect on S1- and acto-S1 ATPase activity.

Smooth muscle myosin phosphatase inhibition and force enhancement by black sponge toxin.

Smooth muscle contraction depends on the state of myosin phosphorylation and hence on the balance of myosin light chain kinase and phosphatase activity. Effects of okadaic acid isolated from black sponge on both enzyme activities and contractility were studied in chemically skinned fibers from guinea pig taenia coli. The toxin strongly inhibits myosin phosphatase and enhances tension development.

Caldesmon-induced inhibition of ATPase activity of actomyosin and contraction of skinned fibres of chicken gizzard smooth muscle.

Caldesmon induces inhibition of MG2+-ATPase activity of actomyosin and relaxation of skinned fibers of chicken gizzard smooth muscle without influencing the level of myosin light chain-1 phosphorylation. Both these effects are reversed by calmodulin at a high molar excess over caldesmon in the presence of Ca2+.

The calmodulin fraction responsible for contraction in an intestinal smooth muscle.

Freeze-dried fibers of smooth muscle from Taenia coli were used to determine the concentration of calmodulin responsible for contraction. About 10% of the total intracellular calmodulin (12.6 mumol/kg wet wt) is directly involved in initiation of smooth muscle contraction.

cGMP-dependent protein kinase decreases calcium sensitivity of skinned cardiac fibers.

Chemically skinned (Lubrol WX) cardiac muscle fibers produce half-maximum isometric tension at pCa 6.18 (pH 6.7) in presence of MgATP (10 mM). After addition of cGMP (5 microM) and cGMP-dependent protein kinase (0.1 microM), the pCa required for half-maximum activation is 5.96, while maximum tension is not affected. Similar shifts in the tension/pCa-relationship have been observed after incubation of skinned cardiac muscle fibers with cAMP of catalytic subunit of the cAMP-dependent protein kinase. The shift in the Ca2+-sensitivity is associated with an increased incorporation of radioactivity into a Mr 28000 band (presumably troponin-I) and a Mr 145000 band.

The influence of P-light chain phosphorylation by myosin light chain kinase on the calcium sensitivity of chemically skinned heart fibres.

Phosphorylation of the P-light chain of myosin might be involved in the regulation of cardiac contractility. Thus an enhanced phosphorylation level of the P-light chain catalyzed by Ca2+-calmodulin-dependent myosin light chain kinase (MLCK) increased significantly the Ca2+ sensitivity of chemically skinned ventricular fibre bundles of the pig. This effect was reversible. Whereas force development at submaximal Ca2+ concentration (pCa 5.5) increased by approximately 50% in the presence of MLCK, maximum tension achieved at maximum Ca2+-concentration (pCa 4.3) was not affected.

A synthetic peptide mimics troponin I function in the calcium-dependent regulation of muscle contraction.

A new technique for treating skinned cardiac muscle fibers has been developed in which troponin I is extracted, giving rise to unregulated fibers. The effect of the 12-residue troponin I peptide on these fibers indicates that this region of troponin I is solely responsible for muscle relaxation (inhibition of force). Furthermore, troponin I peptide-troponin C reconstituted fibers are stable through several contraction-relaxation cycles indicating the peptide can switch binding sites between actin and troponin C. The troponin I peptide can substitute for the native protein as part of the calcium-sensitive molecular switch that controls muscle regulation.

Troponin replacement in permeabilized cardiac muscle. Reversible extraction of troponin I by incubation with vanadate.

Calcium-dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate-free, low-Ca2+ solution (pCa greater than 8), the permeabilized muscle produced 84.8% +/- 20.1% (+/- S.D., n = 98) of the isometric force elicited by high Ca2+ (pCa approximately 4.5) prior to incubation with vanadate. Transfer back to a high Ca2+ solution elicited no additional force (83.2% +/- 18.7% of control force). SDS-PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (greater than 90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.

Striational autoantibodies in myasthenia gravis patients recognize I-band titin epitopes.

Myasthenia gravis (MG) patients develop autoantibodies primarily against the acetylcholine receptor in the motor endplate, but also against intracellular striated muscle proteins, notably titin, the giant elastic protein of the myofibrillar cytoskeleton. Titin antibodies have previously been shown to be directed against a single epitope on the molecule, located at the A-band/I-band junction and referred to as the main immunogenic region (MIR) of titin. By using immunofluorescence microscopy on stretched single myofibrils, we now report that approximately 40% of the sera from 18 MG/thymoma patients and 8 late-onset MG patients with thymus atrophy contain antibodies that bind to a more central I-band titin region. This region consists of homologous immunoglobulin domains and is known to be differentially spliced dependent on muscle type. All patients with I-band titin antibodies also had antibodies against the MIR. Although a statistically significant correlation between the occurrence of I-band titin antibodies and MG severity was not apparent, the results could hint at an initial immunoreactivity to titin's MIR, followed by reactivity along the titin molecule in the course of the disease.

The positive inotropic calcium sensitizer EMD 53998 antagonizes phosphate action on cross-bridges in cardiac skinned fibers.

The diazinone derivative EMD 53998 sensitizes skinned myocardial fibers to Ca2+ and enhances maximal calcium-activated force (pCa = 4.5) by approximately 100%; the EC50 is 10 microM in the absence and about 30 microM in the presence of added inorganic phosphate (10 mM). Although concentrations of added phosphate as low as 0.5 mM inhibit force, at high concentrations of EMD 53998 (> or = 50 microM), phosphate only inhibits at concentrations exceeding 20 mM. These data suggest that the effects of EMD 53998 and phosphate are mutually antagonistic. Importantly, both EMD 53998 and phosphate had similar effects on force generation in troponin I-depleted (Ca(2+)-independent) skinned fibers, thus demonstrating that these compounds are likely to affect cross-bridges directly and not via the Ca(2+)-regulatory system.

The calcium sensitizer EMD 53998 antagonizes phosphate-induced increases in energy cost of isometric tension in cardiac skinned fibres.

We have investigated whether a Ca(2+)-sensitizing substance, the thiadiazinone derivative EMD 53998, can alter the ratio of ATPase activity to force, i.e. the tension cost in skinned fibres of swine cardiac trabecula in which the tension cost was increased by inorganic phosphate. In the presence of 10 mM inorganic phosphate (Pi) and thapsigargin 20 microM, EMD 53998 reduced the energy cost of isometric tension over the entire range of activating Ca2+ concentrations, resulting in a consistent change in slope (approximately 20% decrease) of the ATPase/force relation. We confirmed that in the absence of added phosphate and at maximal Ca2+ activation EMD 53998 had little if any effect on tension cost. We had previously reported that the effects of EMD 53998 and Pi on calcium sensitivity and maximum isometric tension are mutually antagonistic and our new energy data now support the proposal that EMD 53998 functionally antagonizes the effects of Pi on crossbridges. The decrease in the slope of the relation between ATPase and force caused by EMD 53998 may be interpreted to reflect either a decrease in the rate of 'detachment' (g(app)) of crossbridges or an increase in average force per crossbridge, as predicted by classical crossbridge models. Since the Pi release step of the crossbridge cycle is associated with the rate of 'attachment' (f(app)) rather than g(app), we conclude that the decrease in tension cost with EMD 53998 most likely reflects an increased force per crossbridge.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+-cyclic AMP interaction in chemically skinned smooth muscle.

Using guinea-pig taenia coli smooth muscle that is chemically skinned with Triton X-100 to functionally destroy plasmalemma as well as sarcoplasmic reticulum, we demonstrate that cAMP can enhance the extent as well as the rate of relaxation produced by lowering free [Ca2+] in a skinned fiber bundle precontracted with maximal [Ca2+]. However, these actions of cAMP are strongly dependent on the level of free [Ca2+] in the bathing medium, the effect of cAMP being greatly attenuated at higher [Ca2+]. These data support and further extend the results of our previous study indicating that modulations in [Ca2+] can have a strong influence on the ability of cAMP to produce a direct inhibitory effect on the contractile machinery in smooth muscle.

The Ca2+ sensitizer EMD 53998 antagonizes the effect of 2,3-butanedione monoxime on skinned cardiac muscle fibres.

The effects of 2,3-butanedione monoxime (BDM) and 5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-me thy l-3,6-dihydro-2H-1,3,4-thiadiazin-2-one (EMD 53998) on cardiac muscle were studied in skinned muscle fibres from the right ventricle of the porcine heart. BDM decreases the Ca2+ sensitivity (pCa50 for 50% activation) and it exerts a dose-dependent inhibitory effect on force in troponin I (TnI)-depleted (unregulated) cardiac skinned muscle fibres (IC50 approximately 20 mM) thereby mimicking the effect of the TnI inhibitory peptide (cTnI 137-148, corresponding to the cardiac TnI inhibitory region) and that of inorganic phosphate (Pi). This inhibitory action can be antagonized by the calcium-sensitizing cardiotonic thiadiazinone derivative EMD 53998 that increases the IC50 to about 30 mM. In skinned fibres, BDM (10 mM) also increased the ratio of ATPase activity to isometric force (tension cost), whereas EMD 53998 (20 mu M) decreased it. We propose that BDM antagonizes EMD 53998 because both compounds affect the Pi release step of the crossbridge cycle in an antagonistic manner.

Ca2+-sensitizing effect of BM 14.478 on skinned cardiac muscle fibres of guinea-pig papillary muscle.

A concentration-dependent increase in force development was obtained with BM 14.478 (10(-9)-5 X 10(-4) M) in skinned fibres of guinea-pig papillary muscles. Guinea-pig papillary muscles are standard preparations for evaluating inotropic effects and they were also used in the present case for evaluating the positive inotropic effect of BM 14.478. We therefore conclude that a marked calcium-sensitizing effect contributes to the positive inotropic effect obtained with BM 14.478 even at very low concentrations.

Effect of calcium-antagonist and calmodulin-antagonist drugs on calmodulin-dependent contractions of chemically skinned vascular smooth muscle from rabbit renal arteries.

1. Renal arteries from rabbits were chemically skinned by incubation with Triton X-100, and subsequently stored in buffered glycerol. 2. In the presence of Mg-ATP, of EGTA-buffered calcium, and of calmodulin, miniature strips of the skinned arteries developed tension the strength of which was approx. 15-20% of that of viable renal arteries. 3. Tension development was dependent on the concentration of both calcium and calmodulin. 4. The effect of eight vasodilator drugs, the majority of them being "calmodulin antagonists" or "calcium antagonists", on the skinned arteries was assessed. In concentrations up to 10(-3) M, verapamil, D-600, and hydralazine proved to be ineffective, and the same was found with the dihydropyridine derivatives, nifedipine and felodipine, at 0.6 X 10(-3) M and 0.8 X 10(-4) M, respectively, i.e. at saturation in a 9:1 contracting buffer/ethanol mixture (v/v). 5. In a concentration-dependent manner, trifluoperazine, W-7, and fendiline relaxed Ca-calmodulin-induced tension or prevented tension development when given prior to the activation by Ca-calmodulin. However, considerably higher concentrations of the drugs were necessary for half-maximal relaxation than the reported concentrations for half-maximal saturation of hydrophobic binding sites at the calmodulin molecule. 6. These findings suggest that at therapeutic blood levels, the vasodilator properties of calcium antagonists and other direct vasodilators cannot be explained by interference with the binding of myosin light chain kinase to calmodulin.