Ca2+ binding capacity of cytoplasmic proteins from rod photoreceptors is mainly due to arrestin.
(9/77)
Arrestin (also called S-antigen or 48-kDa protein) binds to photoexcited and phosphorylated rhodopsin and, thereby, blocks competitively the activation of transducin. Using Ca2+ titration in the presence of the indicator arsenazo III and 45Ca2+ autoradiography, we show that arrestin is a Ca2(+)-binding protein. The Ca2+ binding capacity of arresting-containing protein extracts from bovine rod outer segments is about twice as high as that of arrestin-depleted extracts. The difference in the Ca2+ binding of arrestin-containing and arrestin-depleted protein extracts was attributed to arrestin. Both, these difference-measurements of protein extracts and the measurements of purified arrestin yield dissociation constants for the Ca2+ binding of arrestin between 2 and 4 microM. The titration curves are consistent with a molar ratio of one Ca2+ binding site per arrestin. No Ca2+ binding in the micromolar range was found in extracts containing mainly transducin and cGMP-phosphodiesterase. Since arrestin is one of the most abundant proteins in rod photoreceptors occurring presumably up to millimolar concentrations in rod outer segments, we suggest that aside from its function to prevent the activation of transducin, arrestin acts probably as an intracellular Ca2+ buffer. (+info)
Biochemical composition of carious dentin and different layers of sound dentin.
(10/77)
The aim of this study was to evaluate the concentrations of calcium (Ca), inorganic phosphate (Pi) and fluoride (F) in carious dentin and in different layers of sound dentin. The samples examined were 52 permanent teeth (26 sound and 26 carious), which were subjected to two experiments to assess the mineral content of: 1) two layers (internal and external) of sound dentin and 2) sound and carious dentin. Ca and Pi were analyzed using a colorimetric method with arsenazo III (C22H18As2N4O14S2) and molybdate reagents, and F was analyzed using a specific electrode. A non-parametric test, the Mann-Whitney test, was used to verify differences between groups. Sound dentin showed a higher concentration of fluoride in the internal layer than in the external layer (P = 0.03), but no inter-layer differences in Ca or Pi concentration were evident. Lower concentrations of Ca, Pi and F were observed in carious dentin than in sound dentin (P < 0.05). The results of this study suggest that the internal layer of sound dentin has a higher fluoride content than the external layer, and that carious dentin has lower concentrations of Ca, Pi and F than sound dentin. (+info)
The influence of chemical agents on the level of ionized [Ca2+] in squid axons.
(11/77)
Squid giant axons injected with either aequorin or arsenazo III and bathed in 3 mM Ca (Na) seawater were transferred to 3 mM Ca (K) seawater and the response of the aequorin light or the change in the absorbance of arsenazo III was followed. These experimental conditions were chosen because they measure the change in the rate of Na/Ca exchange in introducing Ca into the axon upon depolarization; [Ca]o is too low to effect a channel-based system of Ca entry. This procedure was applied to axons treated with a variety of compounds that have been implicated as inhibitors of Na/Ca exchange. The result obtained was that the substances tested could be placed in three groups. (a) Substances that were without effect on Ca entry effected by Na/Ca exchange were: D600 at 10-100 microM, nitrendipine at 1-5 microM, Ba2+ and Mg2+ at concentrations of 10-50 mM, lidocaine at 0.1-10 mM, cyanide at 2 mM, adriamycin at a concentration of 3 microM, chloradenosine at 35 microM, 2,4-diaminopyridine at 1 mM, Cs+ at 45-90 mM, and tetrodotoxin at 10(-7). (b) Substances that had a significant inhibitory effect on Na/Ca exchange were: Mn2+, Cd2+, and La3+ at 1-50 mM, and quinidine at 50 microM. (c) There were also blocking agents and biochemical inhibitors whose action appeared to be the inhibition of nonmitochondrial Ca buffering in axoplasm rather than an inhibition of Na/Ca exchange. These were the general anesthetic l-octanol at 0.1 mM and 1 mM orthovanadate plus apyrase. (+info)
Regional distribution of calcium influx into bursting neurons detected with arsenazo III.
(12/77)
Absorbance changes of the metallochromic indicator arsenazo III were used in conjunction with an array of 100 photodiodes to measure changes in intracellular calcium concentration at many positions simultaneously in identified neurons of the crab stomatogastric ganglion. When stimulated with intrasomatically injected current, several of these neurons showed calcium changes all over the cell, indicating that calcium channels were distributed widely in the neuropil and on the soma. When the membrane potential was allowed to oscillate without stimulation, absorbance oscillations were detected all over the neuropil but not in the soma. A comparison between the membrane potential recorded in the soma and the calcium signal in the neuropil shows that calcium entry followed the slow voltage oscillation with the peak calcium signal detected 50-150 msec after the end of the voltage plateau. (+info)
Calcium entry into voltage-clamped presynaptic terminals of squid.
(13/77)
Voltage-clamp measurements of Ca current and Arsenazo III measurements of intracellular Ca concentration were used to assess Ca ion entry into voltage-clamped presynaptic terminals of squid 'giant' synapses. Depolarization of voltage-clamped terminals filled with Arsenazo III produced absorbance changes consistent with intracellular accumulation of Ca ions. These intracellular Ca transients had a bell-shaped dependence on presynaptic potential and were maximal at approximately -10 mV. Arsenazo III signals recorded from the proximal portion of voltage-clamped presynaptic terminals had a dependence on command potential which was shifted relative to signals recorded from other presynaptic regions. Micro-electrode measurements of presynaptic membrane potential showed that during voltage-clamp depolarizations the proximal region was less depolarized than the rest of the presynaptic terminal. This indicates that voltage-clamped presynaptic terminals may be poorly controlled at their proximal region due to current flow into the adjacent axon. This poor control can cause heterogeneous Ca entry into the presynaptic terminal and thus heterogeneous release of transmitter along the terminal. Application of Ca ions from an extracellular pipette positioned near the distal end of the presynaptic terminal was used to restrict Ca entry to this well-controlled region. Local Ca application decreased the contribution of release from the poorly controlled proximal region to synaptic transfer curves. Presynaptic Ca currents were derived by correcting membrane currents for leakage and capacitive currents and other currents measured in the absence of Ca application. Ca currents measured in this way activated along a sigmoidal time course and did not inactivate for depolarizations as long as 25 ms. Peak Ca currents occurred at approximately -10 mV and inward Ca currents had an apparent 'reversal potential' near +60 mV. Ca channel activation, assessed with tail current measurements, was half-maximal at -13 mV and maximal at +20 mV. Simultaneous measurements of presynaptic Ca currents and Arsenazo III transients revealed a quantitative correspondence between Ca current integrals and Arsenazo III signal amplitude. This suggests that both methods provide reliable measures of Ca ion entry into presynaptic terminals under these conditions. (+info)
Sodium-calcium exchange in the outer segments of bovine rod photoreceptors.
(14/77)
Intact rod outer segments (r.o.s.) isolated from bovine retinas were used to measure net Ca2+ fluxes using the optical Ca2+ indicator Arsenazo III. Ca2+ fluxes were observed, which could change the internal Ca2+ content of isolated r.o.s. by as much as 0.5 mM s-1. The Ca2+ content of isolated intact r.o.s. was strongly dependent on the Na/Ca ratio in the isolation medium, and could be made less than 0.1 mol Ca2+ mol-1 rhodopsin (zero Ca2+ in isolation medium) or up to 7 mol Ca2+ mol-1 rhodopsin (zero Na+ in isolation medium). Ca2+ efflux from r.o.s. rich in Ca2+ was observed only when Na+ was added to the external medium (as opposed to any other alkali cation); in Ca2+-depleted r.o.s. Ca2+ uptake required the presence of internal Na+ and was inhibited selectively by external Na+. These results suggest that Na-Ca exchange across the plasma membrane operated freely in both directions and controlled the internal Ca2+ concentration in r.o.s. Na+-stimulated Ca2+ efflux depended on the external Na+ concentration in a sigmoidal way. This suggests that the simultaneous binding of two Na ions is rate limiting for transport. In Ca2+-depleted r.o.s. and in the absence of external Na+, 1 mol Ca2+ mol-1 rhodopsin (or 3 mM-total Ca2+) could be taken up within 1 min by intact r.o.s. at a free external Ca2+ concentration of about 1 microM. Only part of the internal Ca2+ was available for Na-Ca exchange. The external Na+ and K+ concentration as well as the temperature were factors controlling the accessibility of internal Ca2+ to participate in Na-Ca exchange. Ca2+ fluxes in r.o.s. with a permeabilized plasma membrane but intact disk membranes were very similar to those observed in intact r.o.s.; Na-Ca exchange could operate in both directions across the disk membrane. In addition to Na-Ca exchange, leaky r.o.s. also showed a guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-induced Ca2+ release that was about 1/20 of the rate of Na-Ca exchange. Na-Ca exchange could release 1.5 mol Ca2+ mol-1 rhodopsin from disks as compared with a cyclic-GMP-induced release of 0.15 mol Ca2+ mol-1 rhodopsin. (+info)
Arsenazo III transients and calcium current in a normally non-spiking neuronal soma of crayfish.
(15/77)
Arsenazo III was used to investigate Ca2+ transients in the normally non-excitable soma of the motor giant neurones of the crayfish Procambarus clarkii. Two kinds of regenerative potentials could be obtained depending on membrane potential conditioning: a fast spike after a pre-hyperpolarization to -90 mV and a slow action potential after a pre-depolarization to -50 mV. Only the second of these was accompanied by an Arsenazo III transient. In voltage-clamped, somata injected, with tetraethylammonium chloride, an absorbance change could be obtained by pulsing the membrane potential above -44 mV. The relationship between absorbance change and potential peaked between 0 and +10 mV then fell off to zero at ca. +150 mV. Changes in light absorbance studied using double-pulse protocols suggested that the inactivation of Ca2+ entry was predominantly mediated by the intracellular free Ca2+ concentration. External application of 1 mM-CdCl2 abolished both the absorbance changes and the (Ca2+) inward current. The voltage dependence of this current was similar to that of the absorbance change. For positive membrane potential the current-voltage relationship showed a voltage-dependent conductance property, the origin of which is discussed. (+info)
Calcium levels measured in a presynaptic neurone of Aplysia under conditions that modulate transmitter release.
(16/77)
We have utilized the Ca2+ indicator dye, Arsenazo III, to examine the role of presynaptic Ca2+ concentration in two types of synaptic plasticity observed at the synapses of cell L10 in Aplysia californica; post-tetanic potentiation (p.t.p. - the increased transmitter release which follows high frequency stimulation), and resting membrane potential modulation of release. Intracellular Ca2+ was monitored in the cell body and main neurites of L10 injected with Arsenazo III. Tetanic stimulation caused an increase in intracellular Ca2+ concentration that decayed, after tetanus, with fast and slow time constants which paralleled the time course of decay of p.t.p. When the voltage-sensitive Ca2+ current was reduced by removing external Ca2+ (0 mM-Ca2+, 4 mM-EGTA) or by blocking Ca2+ channels with divalent cation channel blocker (4 mM-Cd2+), tetanic stimulation did not cause increases in Arsenazo absorbance even when Na+ currents were not blocked. This finding suggests that Ca2+ entering the cell through voltage-dependent Ca2+ channels was the major source of Ca2+ which accumulated during the tetanus. Transmitter release is increased when L10 is maintained at a depolarized membrane potential, and is decreased when L10 is hyperpolarized. We found that the base-line Arsenazo absorbance signal in L10 increased when L10 was depolarized from -60 to -40 mV and decreased when L10 was hyperpolarized. This finding supports the idea that the steady-state Ca2+ concentration contributes to the membrane-potential modulation of transmitter release. These results support the idea that transmitter release can be modulated by the residual or resting Ca2+ concentration of the presynaptic cell. (+info)