Arsenazo III used as a calcium ion indicator in mouse oocytes.
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The Ca2+-indicator dye Arsenazo III has been injected into mouse oocytes by intracellular ionophoresis, and the optical absorbance of the injected dye measured using a double-beam microspectrophotometer. The absorbance spectrum shows a broad peak between 565 and 580 nm, with a maximum of 0.020-0.095 in different oocytes. From these values, intracellular dye concentrations were estimated to be 0.1-0.5 mM, although estimated concentrations up to 1.0 mM have been used in later experiments where the dye absorbance was measured only at selected wave-lengths. The transport number for intracellular ionophoresis of the dye has been estimated. The mean value was 0.009 in experiments where the injection current was allowed to cross the cell membrane, but was only 0.0015 when the injection current was balanced by a current of opposite polarity through a second electrode barrel. In oocytes exposed to the mitochondrial uncoupler DNP (2,4-dinitrophenol), the shape of the Arsenazo III absorbance spectrum was found to change. The resulting difference spectra show the peaks at about 610 and 660 nm which are characteristic features of the Arsenazo III Ca2+-difference spectrum. These absorbance changes were not completely reversible on removal of DNP. Changes in the optical absorbance of Arsenazo III have been observed in oocytes injected with Ca2+ by intracellular ionophoresis. Estimated increases of intracellular free [Ca2+], which were of the order of 1 microM and decayed within 30 s, are discussed in relation to the Ca2+-buffering and Ca2+-elimination properties of the oocyte. (+info)
Serotonin increases intracellular Ca2+ transients in voltage-clamped sensory neurons of Aplysia californica.
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Noxious stimulation of the tail of Aplysia californica produces behavioral sensitization; it enhances several related defensive reflexes. This reflex enhancement involves heterosynaptic facilitation of transmitter release from sensory neurons of the reflex. The facilitation is stimulated by serotonin (5-HT) and involves suppression of a 5-HT-sensitive K+ current (the S current). Suppression of the S current broadens the action potential of the sensory neurons and is thought to enhance transmitter release by prolonging entry of Ca2+ in the presynaptic terminals. We now report a component of enhanced Ca2+ accumulation that is independent of changes in spike shape. We have measured intracellular free Ca2+ transients during long depolarizing steps in voltage-clamped sensory neuron cell bodies injected with the Ca2+-sensitive dye arsenazo III. The free Ca2+ transients elicited by a range of depolarizing voltage-clamp steps increase in amplitude by 75% following application of 5-HT. Since it is observed under voltage-clamp conditions, this increase in the free Ca2+ transients is not merely secondary to the changes in K+ current but must reflect an additional mechanism, an intrinsic change in the handling of Ca2+ by the cell. We have not yet determined whether this change in Ca2+ handling reflects an increase in Ca2+ influx, a reduction in intracellular Ca2+ uptake, or a release of Ca2+ from intracellular stores. Regardless of the underlying mechanism, however, it seems possible that the enhancement of Ca2+ accumulation and the reduction in K+ current act synergistically in producing short-term presynaptic facilitation. Alternatively, this additional modulation of Ca2+ by 5-HT might contribute to processes such as classical conditioning or long-term sensitization that may depend on Ca2+. (+info)
Calcium transients studied under voltage-clamp control in frog twitch muscle fibres.
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1. Intracellular calcium transients were recorded from frog twitch muscle fibres in response to voltage-clamped depolarizing pulses, using arsenazo III as an intracellular calcium monitor. The object was to investigate the time- and voltage-dependent characteristics of the coupling process between membrane depolarization and calcium release from the sarcoplasmic reticulum (s.r.)2. To examine the extent to which the T-tubule membrane potential was controlled during clamp pulses, the dye NK 2367 was used as an optical probe of tubular potential. This indicated that the tubular time constant is about 0.6 msec.3. Strength-duration curves were obtained for depolarizing pulses required to give both threshold mechanical contraction and calcium signal. Curves measured in these two ways were closely similar.4. Changes in holding potential altered the strength-duration curve for calcium release so that at more positive holding potentials a shorter pulse was needed to obtain a response for any given pulse amplitude.5. A latency of a few milliseconds was observed between the onset of depolarization and the initial rise of the calcium signal. This became shorter with stronger depolarizations, but approached a minimum at potentials above about +25 mV.6. Subthreshold depolarizations applied before a test pulse increased the size and decreased the latency of the calcium signal. Conditioning hyperpolarizations had opposite effects.7. The rate of build-up of potentiation or depression of response size seen with subthreshold de- and hyperpolarizing conditioning pulses was examined using conditioning pulses of different durations. For both pulses this process showed a time constant of about 3 msec (at 10 degrees C).8. The rate of decay of potentiation or depression was similarly measured, using a gap of variable duration between conditioning and test pulses. For both de- and hyperpolarizing pulses this showed a time constant of about 5 msec (10 degrees C).9. The relationship between conditioning pulse potential, and the size of calcium signal elicited by a following test pulse was non-linear.10. Subthreshold pulses immediately following a brief test pulse affected the size of the calcium signal in a similar way to preceding conditioning pulses.11. The relationship between potential and size of the calcium signal was examined using pulses of 3 and 20 msec duration. With the long pulse the relation was roughly sigmoid, but with the short pulse continued to rise even at strongly positive potentials.12. The results are discussed in terms of a model in which the exponential build-up of a hypothetical coupler in the excitation-contraction (e.-c.) coupling process is presumed to lead to calcium release when a threshold level is exceeded. (+info)
Comparison of isotropic calcium signals from intact frog muscle fibers injected with Arsenazo III or Antipyrylazo III.
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Intact single skeletal muscle fibers were micro-injected with either of the metallochromic indicator dyes Arsenazo III or Antipyrylazo III, and dye-related Ca2+ signals from each were measured during a twitch. In comparison with the Arsenazo III Ca2+ signal, the signal from Antipyrylazo III had three favorable features: (a) it was temporally faster, (b) its spectral dependence agreed with a cuvette calibration, and (c) its kinetic behavior was consistent with a single Ca2+ -dye stoichiometry. It is therefore suggested that the Antipyrylazo III Ca2+ signal is a more accurate monitor of the time course of the underlying myoplasmic free Ca2+ transient and one that may be more reliably calibrated. (+info)
Stoichiometries of arsenazo III-Ca complexes.
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The equilibrium interactions of the metallochromic indicator arsenazo III with calcium at physiological ionic strength and pH were investigated spectrophotometrically and with the aid of a calcium electrode. Evidence suggests the formation of more than one dye-calcium complex. The analysis of data obtained over a 10,000-fold range of dye concentrations concludes that at the concentrations used for in vitro biochemical studies (10--100 microM), arsenazo III absorbance changes in response to calcium binding primarily involve the formation of a complex involving two dye molecules and two calcium ions. At millimolar dye concentrations, typical of physiological calcium transient determinations in situ, a second complex involving two arsenazo III molecules and one calcium ion is additionally formed. A third complex, involving one arsenazo III molecule and one calcium ion, is formed at very low dye concentrations. The results reported here suggest that equilibrium calibration of the dye with calcium cannot be used directly to satisfactorily relate transient absorbance changes in physiological preparations to calcium concentration changes since several stoichiometrically distinct complexes with different absorbances could be formed at different rates. The results of this study do not permit the elucidation of a unique kinetic scheme of arsenazo III complexation with calcium; for this, in vitro kinetic analysis is required. Results of similar analysis of the dye interaction with magnesium are also reported, and these appear compatible with a much simpler model of complexation. (+info)
Sarcoplasmic reticulum calcium release in frog skeletal muscle fibres estimated from Arsenazo III calcium transients.
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Single twitch fibres, dissected from frog muscle, were injected with the metallochromic dye Arsenazo III. Changes in dye-related absorbance measured at 650 or 660 nm were used to estimate the time course of myoplasmic free [Ca2+] following either action potential stimulation or voltage-clamp depolarization (temperature, 15-17 degrees C). The amplitude of the Ca2+ transient decreased when fibres were stretched to sarcomere spacings approaching 4 microns. The effect appeared to be less marked in H2O Ringer than in D2O Ringer, where a reduction of about 40% was observed in going from 3.0 microns to 3.7-3.9 microns. In fibres heavily injected with dye (1.5-2.2 mM-dye) at least 0.1 mM-Ca2+ was complexed with Arsenazo III following a single action potential, implying that at least 0.1 mM-Ca2+ was released from the sarcoplasmic reticulum (s.r.) into the myoplasm. Computer simulations were carried out to estimate the flux of Ca2+ between the s.r. and myoplasm (in fibres containing no more that 0.8 mM-dye). The amounts and time courses of Ca2+ bound to the Ca2+-regulatory sites on troponin and to the Ca2+, Mg2+ sites on parvalbumin were estimated from the free [Ca2+] wave form and the law of mass action. In the computations the total myoplasmic [Ca2+] was taken as the total amount of Ca2+ existing either as free ion or as ion complexed with dye, troponin or parvalbumin. The time derivative of total myoplasmic [Ca2+] was used as an estimate of net Ca2+ flux (release minus uptake) from the s.r. into myoplasm. Rate constants for formation of cation: receptor complex were taken from published values. For the Ca2+-regulatory sites on troponin, three sets of rate constants, corresponding to two values of dissociation constant (0.2 and 2 microM) were used. Each set of three simulations was carried out both with and without parvalbumin. The simulations show that following action potential stimulation, 0.2-0.3 mM-Ca2+ enters the myoplasm from the s.r. The wave form of s.r. Ca2+ release is early and brief compared with the wave form of free [Ca2+]. Neither the selection of troponin rate constants nor the inclusion of parvalbumin has much effect on the shape of the release wave form; the main effect of varying these parameters is to change the magnitude. After the initial, rapid phase of Ca2+ release from the s.r. there is a longer, maintained period of Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS) (+info)
Calcium release and sarcoplasmic reticulum membrane potential in frog skeletal muscle fibres.
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Single twitch fibres were dissected from frog muscle, stretched to a sarcomere spacing greater than or equal to 3.9 micron, then mounted for optical recording. The experiments were carried out at 15-17 degrees C. In some cases D2O Ringer solution was used instead of H2O Ringer solution to reduce movement and any related optical artifacts. Following action potential stimulation, both the amplitude and time course of the change in intrinsic retardation (Baylor & Oetliker's second component, 1975) were found to be approximately independent of wavelength between 480 and 750 nm (D2O Ringer solution). Fibres were injected with the Ca2+-sensitive dye Arsenazo III so that changes in myoplasmic free [Ca2+] could be estimated by measuring changes in dye-related absorbance at 660 nm. The time course of free [Ca2+] was compared with the time course of two other optical signals which have been previously suggested to monitor s.r. (sarcoplasmic reticulum) membrane potential, intrinsic retardation and Nile Blue A fluorescence (Bezanilla & Horowicz, 1975). In D2O Ringer solution the retardation time course was closely similar to that of free [Ca2+] whereas the fluorescence time course was considerably slower. Thus, it is possible that either the retardation signal or Nile Blue A fluorescence (or both) monitors free [Ca2+] rather than s.r. potential. If so, the underlying mechanism which senses Ca2+ must do so very rapidly in the case of retardation and with a delay in the case of Nile Blue A. Changes in Nile Blue A fluorescence were measured in a voltage-clamped fibre (H2O Ringer solution). Only small changes were observed during hyperpolarization or small depolarization whereas relatively large changes were observed near mechanical threshold. These increased e-fold in magnitude every 4-5 mV. This steep voltage dependence, similar to that already shown for intrinsic retardation and [Ca2+], provides additional evidence that Nile Blue A fluorescence monitors a step in excitation-contraction coupling. Theoretical waveforms of s.r. membrane potential were computed using a typical waveform of s.r. Ca2+ release (from Baylor, Chandler & Marshall, 1983) under the assumption that Ca2+ crosses the s.r. membrane as electrical current. Voltage waveforms were calculated using several combinations of electrical parameters for the s.r. membrane. Only certain combinations gave theoretical potential changes similar to the intrinsic retardation or Nile Blue A fluorescence signal.(ABSTRACT TRUNCATED AT 400 WORDS) (+info)
Extracellular ions and excitation-contraction coupling in frog twitch muscle fibres.
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Intracellular calcium transients were recorded from voltage-clamped frog twitch muscle fibres using Arsenazo III. The possible role of extracellular ions in excitation-contraction (e.-c.) coupling was examined using ion substitutions and blocking drugs in the bathing medium. Parameters measured included the Arsenazo response size to a standard depolarizing pulse (5 ms, 0 mV) and the strength-duration curve for threshold Arsenazo signal. Addition of tetrodotoxin (TTX) decreased the response size to small (-30 mV, 5 ms), but not large (+30 mV, 10 ms) depolarizations, probably because of poor voltage clamp of the tubular membrane in the absence of TTX. Clamping TTX-treated fibres with the wave form of a recorded action potential gave an Arsenazo response similar to that elicited by the normal action potential (at 10 degrees C). Complete substitution of sodium (by choline, lithium or Tris) or chloride (by methyl sulphate or maleate) in the bathing solution gave no appreciable changes in the size of the Arsenazo response. Reduction of extracellular free [Ca2+] to low levels using EGTA caused a slight reduction in the calcium signal elicited by the standard depolarization (to 74% after a few hours, and to 62% after 2 days; temperature 5-10 degrees C). The strength-duration curve was unchanged. Arsenazo responses about 75% of the control size could be elicited in high potassium solution (42 mM-K2SO4) by strong (+80 mV, 20 ms) depolarizations, after re-polarizing the fibres to -90 mV for a few minutes. The voltage dependence of activation was shifted to more positive potentials in this solution. Tetraethylammonium (TEA) bromide at a concentration of 20 mM did not alter the Arsenazo signal, whilst 120 mM-TEA reduced the response by 25%. 3,4-diaminopyridine (DAP) reduced the size of the Arsenazo signal at a concentration of 5 mM, and caused spontaneous release of calcium from the sarcoplasmic reticulum (s.r.) in the absence of membrane potential changes. The Arsenazo signal elicited by an action potential was enhanced by 1 mM-DAP, because of prolongation of the action potential, but was depressed by higher concentrations. We conclude that e.-c. coupling does not involve the influx of any external ions into the muscle fibre. If a current flow between the T-tubules and the s.r. is involved in e.-c. coupling, then this is probably carried by an efflux of potassium ions. (+info)