Local control of mitochondrial membrane potential, permeability transition pore and reactive oxygen species by calcium and calmodulin in rat ventricular myocytes.

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaMKII) play important roles in the development of heart failure. In this study, we evaluated the effects of CaM on mitochondrial membrane potential (DeltaPsi(m)), permeability transition pore (mPTP) and the production of reactive oxygen species (ROS) in permeabilized myocytes; our findings are as follows. (1) CaM depolarized DeltaPsi(m) dose-dependently, but this was prevented by an inhibitor of CaM (W-7) or CaMKII (autocamtide 2-related inhibitory peptide (AIP)). (2) CaM accelerated calcein leakage from mitochondria, indicating the opening of mPTP, however this was prevented by AIP. (3) Cyclosporin A (an inhibitor of the mPTP) inhibited both CaM-induced DeltaPsi(m) depolarization and calcein leakage. (4) CaM increased mitochondrial ROS, which was related to DeltaPsi(m) depolarization and the opening of mPTP. (5) Chelating of cytosolic Ca(2+) by BAPTA, the depletion of SR Ca(2+) by thapsigargin (an inhibitor of SERCA) and the inhibition of mitochondrial Ca(2+) uniporter by Ru360 attenuated the effects of CaM on mitochondrial function. (6) CaM accelerated Ca(2+) extrusion from mitochondria. We conclude that CaM/CaMKII depolarized DeltaPsi(m) and opened mPTP by increasing ROS production, and these effects were strictly regulated by the local increase in cytosolic Ca(2+) concentration, initiated by Ca(2+) releases from the SR. In addition, CaM was involved in the regulation of mitochondrial Ca(2+) homeostasis.

Conformational properties of and a reorientation triggered by sugar-water vibrational resonance in the hydroxymethyl group in hydrated beta-glucopyranose.

In this paper, we discuss the conformational properties of the hydroxymethyl group of beta-glucopyranose in aqueous solution and its reorientation mechanism. First, using the values for the hydroxymethyl torsion (O5-C5-C6-O6) angle obtained by our ab initio simulations, we reestimate the experimental ratio of the hydroxymethyl rotamer populations. The reestimated ratio is found to be in agreement with those previously reported in several computational studies, which probably partly explains the discrepancies between theoretical and experimental studies that have been discussed in the literature. Second, our time-frequency analysis on a reorientation in the hydroxymethyl group in an ab initio molecular dynamics trajectory suggests that, before the reorientation, the O6-H6 stretching mode is vibrationally coupled with a proton-accepting first-hydration-shell water molecule, whereas the C6-O6 stretching mode is vibrationally coupled with a proton-donating one. The amount of the total vibrational energy induced by these vibrational couplings is estimated to be comparable to typical values for the potential barriers between hydroxymethyl rotamers. To elucidate the vibrational couplings, we investigate the hydrogen-bonding properties around the hydroxymethyl group during the pretransition period. The implications, validity, and limitation of a possible reorientation mechanism based on these findings are also discussed.

Structural fluctuation and dynamics of ribose puckering in aqueous solution from first principles.

Using the method of ab initio molecular dynamics, we examine the structural fluctuation and the low-frequency dynamics of beta-ribofuranose puckering in aqueous solution. Our analysis suggests that the distance between the anomeric and hydroxymethyl oxygens is a simple relevant geometrical parameter that dynamically correlates with the phase angle in the north region. The time-frequency analysis using the Hilbert-Huang transform also confirms the correlation, and most of the instantaneous frequencies for the phase angle and the above distance are found to be concentrated on the region below about 100 cm(-1). Our analysis of ab initio molecular dynamics trajectories suggests that the molecular origin of the hydration effects on the low-frequency dynamics of beta-ribofuranose puckering is closely related to this correlation and thus primarily attributed to the relatively local interactions among the anomeric and hydroxymethyl oxygens and the surrounding water molecules near them. Additionally, we discuss the difference in the low-frequency dynamics of beta-ribofuranose puckering between two hydroxymethyl rotamers.

Enrichment culture of marine anaerobic ammonium oxidation (anammox) bacteria from sediment of sea-based waste disposal site.

The present study investigates the enrichment of anaerobic ammonium oxidation (anammox) bacteria in the marine environment using sediment samples obtained from a sea-based waste disposal site and discusses the construction of marine anammox bioreactor. Enrichment of bacteria related to Candidatus Scalindua wagneri along with simultaneous removal of nitrite and ammonium ions was observed in the continuous bioreactor culture under a total nitrogen loading rate of 0.4 kg-N m(-3) day(-1).

Protein phosphatase inhibitor-1 augments a protein kinase A-dependent increase in the Ca2+ loading of the sarcoplasmic reticulum without changing its Ca2+ release.

BACKGROUND: An increase in cytosolic protein phosphatases (PPs) de-phosphorylates phospholamban, decreasing the Ca(2+) uptake of the sarcoplasmic reticulum (SR). The effects of PP inhibitors on cellular Ca(2+) handling were investigated. METHODS AND RESULTS: Twitch Ca(2+) transients (CaTs) and cell shortening were measured in intact rat cardiac myocytes, and caffeine-induced Ca(2+) transients (CaffCaTs) and Ca(2+) sparks were studied in saponin-permeabilized cells. Calyculin A augmented isoproterenol-induced increases in CaTs and cell shortening without altering the diastolic [Ca(2+)](i) and twitch [Ca(2+)](i) decay. The protein kinase A catalytic subunit (PKA(cat)) increased the peak of CaffCaTs between 5 and 50 U/ml, and the addition of inhibitor-1 (I-1) augmented the increase. PKA(cat) increased Ca(2+) spark frequency and the addition of I-1 increased it further. PKA(cat) at 50 U/ml amplified the peak and prolonged the duration of Ca(2+) sparks, whereas the addition of I-1 did not alter them. An abrupt inhibition of SR Ca(2+) uptake following exposure to PKA(cat) caused a gradual decrease in Ca(2+) spark frequency, but the addition of I-1 did not accelerate the decline of Ca(2+) spark frequency or CaffCaTs. CONCLUSIONS: Inhibition of PPs augmented the inotropic effect of isoproterenol. Specific inhibition of PP1 could stimulate the Ca(2+) uptake of the SR with less significant effects on the Ca(2+) release.

Different effects of palmitoyl-L-carnitine and palmitoyl-CoA on mitochondrial function in rat ventricular myocytes.

Although mitochondrial oxidative catabolism of fatty acid (FA) is a major energy source for the adult mammalian heart, cardiac lipotoxity resulting from elevated serum FA and enhanced FA use has been implicated in the pathogenesis of heart failure. To investigate the effects of intermediates of FA metabolism [palmitoyl-l-carnitine (Pal-car) and palmitoyl-CoA (Pal-CoA)] on mitochondrial function, we measured membrane potential (DeltaPsi(m)), opening of the mitochondrial permeability transition pore (mPTP), and the production of ROS in saponin-treated rat ventricular myocytes with a laser scanning confocal microscope. Our results revealed that 1) lower concentrations of Pal-car (1 and 5 muM) caused a slight hyperpolarization of DeltaPsi(m) [tetramethylrhodamine ethyl ester (TMRE) intensity increased to 115.5 +/- 5.4% and 110.7 +/- 1.6% of baseline, respectively, P < 0.05] but did not open the mPTP, 2) a higher concentration of Pal-car (10 microM) depolarized DeltaPsi(m) (TMRE intensity decreased to 61.9 +/- 12.2% of baseline, P < 0.01) and opened the mPTP (calcein intensity decreased to 70.7 +/- 2.8% of baseline, P < 0.01), 3) Pal-CoA depolarized DeltaPsi(m) without opening the mPTP, and 4) only the higher concentration of Pal-car (10 muM) increased ROS generation (2',7'-dichlorofluorescein diacetate intensity increased to 3.4 +/- 0.3-fold of baseline). We concluded that excessive exogenous intermediates of long-chain saturated FA may disturb mitochondrial function in different ways between Pal-car and Pal-CoA. The distinct mechanisms of the deteriorating effects of long-chain FA on mitochondrial function are important for our understanding of the development of cardiac diseases in systemic metabolic disorders.

Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes.

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.