Effect of fura-2 on action potential-stimulated calcium release in cut twitch fibers from frog muscle.

Cut fibers (striation spacing, 3.6-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14-15 degrees C. One or both of the Ca indicators fura-2 and purpurate-3,3' diacetic acid (PDAA) were introduced into the optical recording site by diffusion from the end pools. Sarcoplasmic reticulum (SR) Ca release was elicited by action potential stimulation. With resting [fura-2] = 0 mM at the optical site, the [Ca] transient measured with PDAA was used to estimate SR Ca release (Baylor, S.M., W.K. Chandler, and M.W. Marshall. 1983. Journal of Physiology. 344:625-666). With resting [fura-2] > 0 mM, the contribution from Ca complexation by fura-2 was added to the estimate. When resting [fura-2] was increased from 0 to 0.5-2 mM, both the amount of SR Ca release and the maximal rate of release were increased by approximately 20%. These results are qualitatively similar to those obtained in intact fibers (Baylor, S.M., and S. Hollingworth. 1988. Journal of Physiology. 403:151-192; Hollingworth, S., A. B. Harkins, N. Kurebayashi, M. Konishi, and S. M. Baylor. 1992. Biophysical Journal. 63:224-234) and are consistent with a reduction of Ca inactivation of SR Ca release produced by 0.5-2 mM fura-2. With resting [fura-2] > or = 2 mM, the PDAA [Ca] transient was reduced to nearly zero and SR Ca release could be estimated from delta [Cafura-2] alone. When resting [fura-2] was increased from 2-4 to 5-6 mM, both the amount of SR Ca release and the maximal rate of release were decreased by approximately half, consistent with a possible reduction of Ca-induced Ca release (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophysical Journal. 60:867-873) or a possible pharmacological effect of fura-2.

Reduction of calcium inactivation of sarcoplasmic reticulum calcium release by fura-2 in voltage-clamped cut twitch fibers from frog muscle.

Cut fibers from Rana temporaria and Rana pipiens (striation spacing, 3.9-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14 degrees C. The Ca indicator purpurate-3,3' diacetic acid (PDAA) was introduced into the end pools and allowed to diffuse into the optical recording site. When the concentration at the site exceeded 2 mM, step depolarizations to 10 mV were applied and the [Ca] transient measured with PDAA was used to estimate Ca release from the sarcoplasmic reticulum (SR) (Baylor, S. M., W. K. Chandler, and M. W. Marshall. 1983. Journal of Physiology. 344:625-666). With depolarization, the rate of SR Ca release increased to an early peak and then rapidly decreased several-fold to a quasi-steady level. The total amount of Ca released from the SR at the time of peak rate of release appeared to be independent of SR Ca content, consistent with the idea that a single activated channel might pass, on average, a fixed number of ions, independent of the magnitude of the single channel flux. A possible explanation of this property is given in terms of locally induced Ca inactivation of Ca release. The solution in the end pools was then changed to one with PDAA plus fura-2. SR Ca release was estimated from the [Ca] transient, as before, and from the delta [Cafura-2] signal. On average, 2-3 mM fura-2 increased the quasi-steady level of the rate of SR Ca release by factors of 6.6 and 3.8, respectively, in three fibers from Rana temporaria and three fibers from Rana pipiens. The peak rate of release was increased in five of the six fibers but to a lesser extent than the quasi-steady level. In all fibers, the amplitude of the free [Ca] transient was markedly reduced. These increases in the rate of SR Ca release are consistent with the idea that Ca inactivation of Ca release develops during a step depolarization to 10 mV and that 2-3 mM fura-2 is able to reduce this inactivation by complexing Ca and thereby reducing free [Ca]. Once the concentration of fura-2 becomes sufficiently large, a further increase reduces the rate of SR Ca release. On average, 5-6 mM fura-2 increased the quasi-steady rate of release, compared with 0 mM fura-2, by 6.5 and 2.9, respectively, in four fibers from Rana temporaria and three from Rana pipiens.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of thyroid hormones and their analogues on the mitochondrial calcium transport activity.

In this paper the authors studied the effects of thyroid hormones and their structural analogues on the mitochondrial calcium transport activities. The thyroid hormones, 3,5,3' L-triiodothyronine (LT3) and 3,5,3'5' L-tetraiodothyronine (LT4) at physiological intracellular concentrations between 7.2 and 9 nM, decouple total Ca++ transport, as well as inhibit the passive transport of Ca++, either due to oxidation of pyruvate, malate or succinate or after inhibition with rotenone. The optical isomers 3,5,3' D-triiodothyronine (DT3) and 3,5,3',5' D-tetraiodothyronine (DT4) are less effective at all the used concentrations. Furthermore the structural analogues 3,3',5' L-triiodothyronine (LrT3), 3,5-dicloro, 3',5' L-diiodothyronine (LDiClT2) and 3,5 L-diiodothyronine (LT2) furnished even less effects on the same activities. The effect of the thyroid hormones and of their structural analogues has revealed that the mitochondrial calcium transport may be influenced both by a stereospecific interaction between hormones and protein ligands and by a lipophilic chaotropic action on the mitochondrial membranes lipids. In this context it is interesting to consider that both thyroid hormones and Ca++ transport activity are interacting with the energetic metabolism by means of phosphorylation and substrate oxidation mechanism.

Low concentrations of A23187 increase calcium uptake by cardiac sarcoplasmic reticulum.

The divalent cation ionophore A23187 at a concentration of 1 nM produced an increased rate of oxalate-supported calcium uptake by isolated cardiac sarcoplasmic reticulum as determined by absorbance changes of the calcium-sensitive dye murexide. Addition of a higher concentration of A23187 (0.1 microM) produced a decreased rate of calcium uptake. Measurement of the time during which ATPase was activated by calcium addition also suggested an increased rate of calcium uptake in the presence of 1 nM A23187 and an inhibition of calcium uptake at a higher concentration of the ionophore (0.1 microM). Ca2+-stimulated ATPase activity and incorporation of 32Pi from [gamma-32P]ATP into sarcoplasmic reticular proteins were increased by A23187 at concentrations of 1 nM or greater. An increased coupling of calcium uptake to ATP hydrolysis was observed at 1 nM A23187, while concentrations of the ionophore greater than or equal to 10 nM produced a decreased coupling. Addition of an inhibitor of cyclic AMP-dependent protein kinase decreased the rate of calcium uptake, and this inhibition was reversed in a concentration-dependent manner by 0.01-1 nM A23187. These data suggest that A23187 can activate a mechanism involving the calcium-dependent phosphorylation of protein that may regulate the activity of the calcium uptake system of the sarcoplasmic reticulum. These observations appear to provide an explanation for some of the contractile effects of A23187 in intact cardiac muscle that suggest that treatment with the ionophore results in an increased sequestration of calcium from the cytoplasm.

Generation of free radical metabolites and superoxide anion by the calcium indicators arsenazo III, antipyrylazo III, and murexide in rat liver microsomes.

At the concentrations usually employed as a Ca2+ indicator, arsenazo III undergoes a one-electron reduction by rat liver microsomes to produce an azo anion radical as demonstrated by electron spin resonance spectroscopy. Either NADH or NADPH can serve as a source of reducing equivalents for the production of this free radical by rat liver microsomes. The steady state concentration of the azo anion radical is proportional to the square root of the protein concentration, suggesting that the radical decays through a nonenzymatic second order process. The steady state concentration of the azo anion radical is not altered in the presence of metyrapone or CO, and is decreased in the presence of NADP+ or p-hydroxymercuribenzoate. These observations suggest that the formation of arsenazo III anion radical is mediated through NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Under aerobic conditions, addition of arsenazo III to rat liver microsomes produces an increase in electron flow from NAD(P)H to molecular oxygen, generating both superoxide anion and hydrogen peroxide. The steady state concentration of the azo anion radical, but neither oxygen consumption nor superoxide anion formation, is enhanced by calcium and magnesium, suggesting an enhanced azo anion radical-stabilization by complexation with the metal ions. Accordingly, the arsenazo III anion radical signal is abolished in the presence of paramagnetic metal ions (Fe3+, Gd3+, and Ni2+) and enhanced in the presence of other diamagnetic metal ions (La3+). Antipyrylazo III is less effective than arsenazo III in increasing superoxide anion formation by rat liver microsomes, and gives a much weaker ESR spectrum of an azo anion radical. Murexide is reduced to the monodehydro-5,5'-iminobarbituric acid radical by rat liver microsomes, and its efficiency as a superoxide anion generator is intermediate between arsenazo III and antipyrylazo III.

Direct measurements of the dextran-dependent calcium uptake by rat peritoneal mast cells.

The metallochromic indicator murexide has been used to monitor calcium concentration changes during the dextran-induced, phosphatidylserine-dependent degranulation of rat peritoneal mast cells. The dextran-induced Ca2+-uptake showed an absolute dependence on the presence of phosphatidylserine. The extent of Ca2+-uptake increased with phosphatidylserine in a concentration-dependent manner. At 25 degrees C the half-life of the uptake process equalled 35 +/- 5 s. Exposure of the mast cells to dextran in the presence of Ca2+, but in the absence of phosphatidylserine, desensitized the cells. The subsequent addition of phosphatidylserine failed to restore the Ca2+-uptake activity. However, the Ca2+-ionophore A23187 did promote Ca2+ uptake by the cells without PS.

Interaction of metallochromic indicators with calcium sequestering organelles.

Examples are presented of the interaction between cell organelles and metallochromic indicators used in the measurement of ionized Ca2+. Sarcoplasmic reticulum was found to sequester murexide type indicators along with Ca2+ in the presence of ATP, but not to sequester arsenazo III and antipyrylazo III. The presence of a permeable anion suppresses the sequestration of murexide type indicators by the sarcoplasmic reticulum. In the presence of ruthenium red, both rat liver and beef heart mitochondria release sequestered Ca2+ with arsenazo III, but not with murexide.

Dansylaziridine-labeled troponin C. A new fluorescent "physiological" probe for calcium regulation by sarcoplasmic reticulum.

Dansylaziridine-labeled troponin C (TnCDANZ) undergoes a greater than 2-fold fluorescence enhancement with Ca2+ binding to the Ca2+-specific regulatory sites of troponin C. Hence, TnCDANZ serves as a very good indicator of Ca2+ in the range of 3.0 to 70 micron. Ca2+ uptake and release by sarcoplasmic reticulum can be easily and accurately monitored by the fluorescence changes in TnCDANZ. In this manner, we can monitor directly the removal of Ca2+ from troponin C by the sarcoplasmic reticulum. Thus, this reaction represents a useful analogue ot the Ca2+ "on-off" process in muscle.