Abnormal Ca2+ release from cardiac sarcoplasmic reticulum in tachycardia-induced heart failure. (1/77)

OBJECTIVE: In heart failure, little information is available as to the Ca2+ release function of sarcoplasmic reticulum (SR), which plays a major role in cardiac contractile function. Here, we assessed the rapid kinetics of drug-induced Ca2+ release from cardiac SR in combination with a measurement of ryanodine binding in heart failure. METHODS: The SR vesicles were isolated from dog left ventricular (LV) muscles (normal (N), n = 10; pacing induced heart failure (HF), n = 10). The time course of SR Ca2+ release was continuously monitored by a stopped-flow apparatus using arsenazoIII as a Ca2+ indicator, and Ca2+ uptake and [3H]ryanodine binding assays were done using a filtration method. RESULTS: The amount of Ca2+ uptake was reduced in HF to 55% of N (P < 0.05). Even the more marked and earlier appeared decrease was seen in the rate constant and the initial rate of polylysine (PL; a specific release trigger)-induced Ca2+ release (P < 0.05). However, the PL concentration dependency of the initial rate shifted towards lower concentrations of PL in HF than in N ([PL] at half maximum stimulation = 0.13 vs. 0.35 microM). The [3H]ryanodine binding assay revealed a lower Bmax (pmol/mg) in HF than in N (0.91 +/- 0.19 vs. 2.64 +/- 0.59, P < 0.05), but no difference in Kd (nM) (0.95 +/- 0.29 vs. 0.90 +/- 0.11, P = n.s.). The [PL] dependency on the enhancement of [3H]ryanodine binding again showed a shift towards lower [PL] in HF than in N. CONCLUSIONS: In pacing-induced heart failure, the Ca2+ releasing function of SR is disturbed, which may result in an intra-cellular Ca2+ transient that was slowed down.  (+info)

Calcium release from Synechocystis cells induced by depolarization of the plasma membrane: MscL as an outward Ca2+ channel. (2/77)

Cells of the cyanobacterium Synechocystis sp. PCC 6803 are equipped with a mechanosensitive ion channel MscL that is located in their plasma membrane. However, the exact function of the channel in this freshwater cyanobacterium is unknown. This study shows that cells of Synechocystis are capable of releasing Ca(2+) in response to depolarization of the plasma membrane by the K(+) ionophore valinomycin in the presence of K(+) or by tetraphenylphosphonium (TPP(+)). A fluorescent dye, diS-C(3)-(5), sensitive to membrane potential and the metallochromic Ca(2+) indicator arsenazo III were used to follow the plasma membrane depolarization and the Ca(2+) release, respectively. The Ca(2+) release from wild-type cells was temperature-dependent and it was strongly inhibited by the Ca(2+) channel blocker verapamil and by the mechanosensitive channel blocker amiloride. In MscL-deficient cells, Ca(2+) release was about 50 % of that from the wild-type cells. The mutant cells had lost temperature sensitivity of Ca(2+) release completely. However, verapamil and amiloride inhibited Ca(2+) release from these cells in same manner as in the wild-type cells. This suggests the existence of additional Ca(2+) transporters in Synechocystis, probably of a mechanosensitive nature. Evidence for the putative presence of intracellular Ca(2+) stores in the cells was obtained by following the increase in fluorescence intensity of the Ca(2+) indicator chlortetracycline. These results suggest that the MscL of Synechocystis might operate as a verapamil/amiloride-sensitive outward Ca(2+) channel that is involved in the plasma-membrane depolarization-induced Ca(2+) release from the cells under temperature stress conditions.  (+info)

Single stable reagent (Arsenazo III) for optically robust measurement of calcium in serum and plasma. (3/77)

We describe a method based on a single stable reagent for the determination of calcium in serum and plasma with use of Arsenazo III, 200 mumol/L in 50 mmol/L 1,4-piperazinediethanesulfonic acid (PIPES) buffer. The method showed significant positive interference in plasma at pH less than 6.6 because of the precipitation of fibrinogen, which was eliminated by increasing the pH to 6.8. The assay showed no interference from as much as 600 mumol of bilirubin and 12 g of hemoglobin per liter when applied in a simple monochromatic procedure at 660 nm. The standard curve for calcium was linear from 0 to 5.0 mmol/L. Addition of Intralipid at concentrations greater than 3 g/L demonstrated positive interference, which could be eliminated by using a 700-nm blanking wavelength. The procedure showed good agreement with all-method mean values from two external quality-control schemes.  (+info)

Measuring albumin and calcium in serum in a dual test with the Hitachi 704. (4/77)

We describe a method for simultaneously determining albumin, by using bromcresol purple, and calcium, by using Arsenazo III, in the same analytical cuvette on the Hitachi 704. Both assays agree well with accepted procedures. The standard curves for the albumin and calcium assays are linear from 0 to 60 g/L and 0 to 5.0 mmol/L, respectively. Calibration is stable for 7 days with use of open reagent in the instrument. Both assays are unaffected by hemoglobin less than or equal to 5 g/L and Intralipid less than or equal to 4 g/L; calcium is unaffected by bilirubin less than or equal to 600 mumol/L.  (+info)

Study on the binding of Arsenazo-TB to human serum albumin by Rayleigh light scattering technique and FT-IR. (5/77)

The interaction between Arsenazo-TB and human serum albumin (HSA) was studied by Rayleigh light scattering (RLS) technique and Fourier transformed IR (FT-IR). The binding parameters of Arsenazo-TB with HSA were studied at different temperature of 288, 298, 308, 318 K under the optimum conditions. It is indicated by the Scatchard plots that the binding constant K decreased from 5.03 x 10(7) to 7.13 x 10(6) and the maximum binding number N reduced from 53 to 36 with the increasing of the temperature. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction. The free energy change deltaG0, the enthalpy change deltaH0 and the entropy change deltaS0 of 288 K were calculated to be -42.46 kJ/mol, -49.17 kJ/mol and 318.15 J/mol K, respectively. The alterations of protein secondary structure in the presence of Arsenazo-TB in aqueous solution were quantitatively calculated from FT-IR spectroscopy with reductions of alpha-helix from 57% to 40% and with increases of beta-sheet from 36% to 39%, beta-turn from 7% to 21%.  (+info)

Tracking of proton flow during transition from anaerobiosis to steady state. 2. Effect of cation uptake on the response of a hydrophobic membrane bound pH indicator. (6/77)

1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification). 2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization. 3. The comparison of the bromothymol blue response with the arsenazo III, 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b) phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4. 4. Although bromothymol blue always behaves as a membrane-bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane-potential-determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer. 5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported.  (+info)

Electrogenic Na(+)-Ca2+ exchanger, the link between intra- and extracellular calcium in the Limulus ventral photoreceptor. (7/77)

1. Limulus ventral photoreceptors were injected with Arsenazo III and the internal change in the calcium concentration, [Ca2+]i, was measured under voltage clamp conditions. It is shown that in response to a light flash the rising phase of the [Ca2+]i is independent of the clamp voltage, Vm. This observation is contrary to other results reported in the literature. Experiments are reported that resolve this contradiction (see paragraph 4). 2. The relaxation of the [Ca2+]i after a bright light flash was observed to have a fast and slow phase. A function consisting of the sum of an exponential and a ramp was fitted to the relaxation. The fast phase, characterized by the time constant of the exponential, was observed not to depend on Vm, while the slow phase, characterized by the slope of the ramp, was strongly dependent on Vm. Furthermore the slope of the slow phase is shown to depend on the external Na+ concentration, but not the time constant of the fast phase. 3. In the dark the [Ca2+]i was observed to increase when the cell was depolarized and to decrease when the cell was hyperpolarized. This observation was more pronounced when the cell was continuously illuminated. 4. When the cell was clamped to a depolarizing voltage before illumination of the cell, the maximum of the calcium indicator signal was observed to depend on how long the cell had been clamped before applying the light stimulus. This experiment resolves the contradiction mentioned in paragraph 1. 5. The results presented here are consistent with the interpretation that a Na(+)-Ca2+ exchanger with a stoichiometry greater than 2:1 is the predominant link between intra- and extracellular calcium. Secondly that the light-induced intracellular calcium increase comes from a release by intracellular stores. Finally a measurable uptake of calcium occurs after a light-induced release, possibly by the internal calcium stores. The two-phase recovery of [Ca2+]i after a light flash is interpreted as being a calcium uptake by the internal stores, the fast phase, and removal by the electrogenic Na(+)-Ca2+ exchanger, the slow phase.  (+info)

A heart mitochondrial Ca2(+)-dependent pore of possible relevance to re-perfusion-induced injury. Evidence that ADP facilitates pore interconversion between the closed and open states. (8/77)

The permeability properties of a putative Ca2(+)-activated pore in heart mitochondria, of possible relevance to re-perfusion-induced injury, have been investigated by a pulsed-flow solute-entrapment technique. The relative permeabilities of [14C]mannitol, [14C]sucrose and arsenazo III are consistent with permeation via a pore of about 2.3 nm diameter. Ca2+ removal with EGTA induced pore closure, and the mitochondria became 'resealed'. The permeability of the unresealed mitochondria during resealing was markedly stimulated by 200 microM-ADP, and the relative permeabilities to solutes of different size were stimulated equally, indicating an increase in open-pore number, rather than an increase in pore dimensions. This is paradoxical, since ADP also stimulated the rate of resealing. The rate of EGTA-induced resealing was also stimulated by the Ca2+ ionophore A23187, which indicates that the rate of removal of matrix free Ca2+ is limiting for pore closure. An explanation for the paradox is suggested in which ADP facilitates pore interconversion between the closed and open states in permeabilized mitochondria, and pore closure in Ca2(+)-free mitochondria occurs much faster than previously thought.  (+info)

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