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Journal of Cellular and Comparative Physiology 02/2005; 51(3):425 - 438.
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L J Mullins
Annals of the New York Academy of Sciences 02/1991; 625:841-4. · 3.15 Impact Factor
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L J Mullins
Annals of the New York Academy of Sciences 02/1991; 639:96-8. · 3.15 Impact Factor
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ABSTRACT: Squid giant axons were injected simultaneously with Ca indicators Fura-2 and aequorin. Fura-2 was calibrated in situ by measuring fluorescence at 510 nm upon UV excitation at 340 nm, 360 nm, and 380 nm with a time-sharing multiple wavelength spectrofluorimeter. Limiting values for dye fluorescence were obtained by allowing a massive load of Ca to enter the axon with the aid of procedures such as prolonged depolarization in the presence of CN (for saturation) and by sequestration of all Ca present in the axoplasm accomplished with injection of EGTA into the axon (for a zero-Ca signal). The average intracellular Ca concentration obtained with Fura-2 was 184 nM. The sensitivity of Fura-2 to intracellular Ca is at least as great as that of aequorin, thus permitting its use in the characterization of Ca homeostasis mechanisms such as Na-Ca exchange. It was found, however, that for voltage-clamp experiments requiring an internal current electrode, Fura-2 is not a convenient Ca probe because electrode reactions in the axoplasm denature the dye, thereby restricting its use in characterization of Ca movements associated with electrically induced changes in membrane potential. A comparison of aequorin luminescence with Fura-2 fluorescence demonstrated that light output by aequorin is linear with intracellular Ca concentrations up to values of 750 nM, changing to a square law relationship from 750 nM up to 10 microM Ca.
Annals of the New York Academy of Sciences 02/1991; 639:112-25. · 3.15 Impact Factor
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Society of General Physiologists series 02/1987; 42:65-75.
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ABSTRACT: Squid axons were injected with arsenazo III and treated with sea water containing compounds usually classified as general anesthetics, (pentanol-decanol and a variety of hydrocarbons and their derivatives). Such treatment led to an increase in absorbance by arsenazo III at wavelengths sensitive to [Ca]i. The effect was independent of the presence or absence of Ca++ in sea water and it was not modified by substances that release Ca from internal stores. The effect was easily reversible. In axons injected with phenol red or impaled with a glass electrode sensitive to H+, a similar treatment led to an alkalinization that was also readily reversible. Both Ca release and the change to an alkaline pH had identical time courses. The dose required for action by all of the chemical agents studied could be predicted from a knowledge of their fractional saturation in sea water, i.e. from their thermodynamic activity. For compounds with 8-10 carbon atoms, Ca-release effects can occur at concentration less than those necessary to block either conduction or Na/Ca exchange. A special chemical agent was octylamine, which induced a marked rise in pHi and in addition its nonionic form produced the typical Ca release associated with general anesthetics.
Biophysical Journal 08/1986; 50(1):11-9. · 3.65 Impact Factor
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ABSTRACT: The level of intracellular Ca in squid axons (both ionized and total Ca) was studied as a function of the experimental variables [Na]i, [Na]o, pHi, cyanide, and depolarization. Ionized Ca was measured by following the light emission of aequorin while total Ca was measured by the atomic absorption analysis of samples of axoplasm. Aequorin glow is known to be increased either by the application of Nao-free solutions or by depolarization produced by external solutions containing greater than normal K concentrations. The present results show that if [Na]i is low, the depolarization that is brought about by solutions with elevated [K] leads to a resting light emission that is decreased rather than increased, as is the case when [Na]i is high. In axons where [Na]i is varied, a comparison of the increments in light emission produced by the application first of Na-free and then of high-K solutions shows that they have an identical dependence on [Na]i, with a half-activation of Ca entry produced by an [Na]i of 25-30 mM. Changes in pHi affect the aequorin signal produced by depolarization, with acidification reducing and alkanization increasing the response. Cyanide did not greatly affect the size of the signal resulting from either Nao removal or that from depolarization.
The Journal of General Physiology 02/1986; 87(1):143-59. · 3.84 Impact Factor
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ABSTRACT: 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.
The Journal of General Physiology 07/1985; 85(6):789-804. · 3.84 Impact Factor
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ABSTRACT: Intact squid axons were injected with aequorin and bathed in 3 mM Ca seawater (a concentration close to that of squid blood). Sodium and potassium currents were pharmacologically blocked and repetitive voltage-clamp pulses of a duration of 1.5 ms were applied (to simulate the duration of an action potential) at amplitudes of +30 to +90 mV and at frequencies of 100/s. In a very fresh axon (low internal Na concentration) no detectable change in aequorin glow resulted from this treatment, whether the axons were in Na-containing or in Na-free seawater. In axons subjected to modest Na loading, repetitive voltage-clamp pulsing did not result in an increased aequorin glow when the pulses were delivered in Na seawater, whereas in Na-free seawater there was an easily measurable increase in aequorin light emission during repetitive pulsing. The increase in aequorin photons emitted per voltage-clamp pulse was e-fold for 22 mV of depolarization, and the process showed no signs of saturating at pulse amplitudes of +180 mV (i.e., at a membrane potential close to ECa). The aequorin light emission per voltage-clamp pulse increased linearly with pulse duration (at constant amplitude).
Proceedings of the National Academy of Sciences 04/1985; 82(6):1847-51. · 9.68 Impact Factor
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ABSTRACT: Squid giant axons were injected with aequorin or arsenazo III and impaled with a Ca-sensing electrode. The light output of aequorin or the spectrophotometer output when measuring arsenazo was compared with the voltage output of the electrode when the squid axon was depolarized with high-K solutions, when the seawater was made Na-free, or when the axon was tetanized for several minutes. The results from these treatments were that the optical response rose (as much as 50-fold) with all treatments known to increase Ca entry, while the electrode remained unaffected by these treatments. If axons previously subjected to Ca load are treated with electron-transport poisons such as CN, it is known that [Ca]i rises after a time necessary to deplete ATP stores. In such axons one expects a rise of [Ca]i in axoplasm which does not necessarily have to be uniform although the source of such Ca is the mitochondria and these are uniformly distributed in axoplasm. Under conditions of CN application, the optical signals from aequorin or arsenazo and Ca electrode output do rise together when [Ca]i is high, but there is a region of [Ca]i concentration where aequorin light output or arsenazo absorbance rises while electrode output does not. Axons not loaded with Ca but injected with apyrase and vanadate have mitochondria that still retain some Ca and this can be released by CN in a truly uniform manner. The results show that such a release (which is small) can be readily measured with aequorin, but again the Ca electrode is insensitive to such [Ca]i change.
Biochimica et Biophysica Acta 01/1985; 805(4):393-404. · 4.66 Impact Factor
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ABSTRACT: Squid giant axons were impaled with electrodes to measure pNai, pHi, Em, and were injected with either aequorin or arsenazo III to measure [Ca]i or with phenol red to measure [H]i. Depolarization of such axons with elevated [K] in sea water leads to a Ca entry that is a function of [Ca]o, [Na]i, and [H]i. With saturating [Na]i half-maximal Ca entry is produced by a [Ca]o of 0.58 mM. With saturating [Ca]o, depolarization produced by 450 mM-K+ leads to half-maximal Ca entry when [Na]i is 25 mM; entry is virtually undetectable if [Na]i is 18 mM. If [Ca]o is 50 mM, Ca entry upon depolarization as measured with aequorin is phasic with a rapid phase of light emission and a plateau; Ca entry as measured with arsenazo III shows no such phasic behaviour, absorbance vs. time is a square wave that closely follows the depolarization vs. time trace. Both detectors of [Ca]i show a square-wave response if [Ca]o is 3 mM. The introduction of 2 mM-CN into the sea water bathing the axon does not affect the response to depolarization nor does the destruction of most of the ATP in the axon following the injection of apyrase. If axons are microinjected with phenol red rather than arsenazo, the entry of Ca produces an acidification in the peripheral parts of the axoplasm. Other experiments measuring [Ca]i show that Ca entry is strongly inhibited by a decrease in pHi. Making sea water alkaline with pH buffers scarcely affects the Ca entry induced by depolarization; making axoplasm alkaline by adding NH4+ to sea water greatly enhances Ca entry by Na/Ca exchange and also enhances the ability of axoplasmic buffers to absorb Ca.
The Journal of Physiology 06/1983; 338:295-319. · 4.72 Impact Factor
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ABSTRACT: Squid giant axons were injected with aequorin and then treated with seawater containing 50 mM Ca and 100-465 mM K+. Measurements of light production suggested a phasic entry of Ca as well as an enhanced steady-state aequorin glow. After a test K+ depolarization, the aequorin-injected axon was stimulated for 30 min in Li seawater that was Ca-free, a procedure known to reduce [Na]i to about one-half the normal concentration. Reapplication of the elevated K+ test solution now showed that the Ca entry was virtually abolished by this stimulation in Li. A subsequent stimulation of the axon in Na seawater for 30 min resulted in recovery of the response to depolarization by high K+ noted in a normal fresh axon. In axons first tested for a high K+ response and then stimulated in Na seawater for 30 min (where [Na]i increases approximately 30%), there was approximately eight fold enhancement in this response to a test polarization. Axons depolarized with 465 mM K seawater in the absence of external Ca for several minutes were still capable of producing a large phasic entry of Ca when [Ca]0 was made 50 mM, which suggests that it is Ca entry itself rather than membrane depolarization that produced inactivation. Responses to stimulation at 60 pulses/s in Na seawater containing 50 mM Ca are at best only 5% of those measured with high K solutions. The response to repetitive stimulation is not measurable if [Ca]o is made 1 mM, whereas the response to steady depolarization is scarcely affected.
The Journal of General Physiology 01/1982; 78(6):683-700. · 3.84 Impact Factor
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ABSTRACT: Free calcium in the range of 20--60 nM can be analyzed in single nerve fibers of the squid. Analytical methods used where: (1) the photoprotein aequorin, and (2) Arsenazo III, a dye which undergoes a spectral transformation when it interacts with Ca. The free calcium concentration of fresh squid axoplasm is 20--50 nM. Most cell calcium is buffered by organelles; only about 0.1% is ionized. Smooth endoplasmic reticulum appears to be a significant calcium buffer, and also, in some cases, the mitochondria located immediately adjacent to the membrane.
Scanning electron microscopy 02/1980;
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ABSTRACT: Aequorin was microinjected into squid giant axons, the axons were stimulated, and the change in light emission was followed. This response was compared with that found when the axon, in addition to being microinjected with aequorin, is also injected with the dye phenol red. Large concentrations of phenol red injected into axons result in a high probability that photons emitted by aequorin, when it reacts with Ca in the core of the axoplasm, will be absorbed before they escape from the axon; photons produced by the aequorin reaction at the periphery of the axoplasm are much less likely to be absorbed. This technique thus favors observing changes in Cai taking place in the periphery of the axon. Stimulation in 50 mM Ca seawater of an aequorin-phenol red-injected axon at 180 s-1 for 1 min produces a scarcely detectable change in Cai; the addition of 2 mM cyanide (CN) to the seawater produces an easily measureable increase in Cai, suggesting that mitochondrial buffering in the periphery is substantial. Making the pH of the axoplasm of a normal axon alkaline with 30 mM NH4+ -50 mM Ca seawater, reduces the resting glow of the axon but results in an even more rapid increase in Cai with stimulation. In a phenol red-injected axon, this treatment results in a measureable response to stimulation in the absence of CN.
The Journal of General Physiology 10/1979; 74(3):393-413. · 3.84 Impact Factor
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Quarterly Reviews of Biophysics 09/1979; 12(3):371-460. · 10.09 Impact Factor
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ABSTRACT: Axons freshly dissected from living specimens of the tropical squid Dorytheutis plei have a calcium content of 68 mumol/kg of axoplasm. Fibers stimulated at 100 impulses/s in 100 mM Ca seawater increase their Ca content by 150 mumol/kg.min; axons placed in 3 Ca (choline) seawater increase their Ca content by 12 mumol/kg.min. Axons loaded with 0.2--1.5 mmol Ca/kg of axoplasm extruded Ca with a half time of 15--30 min when allowed to recover in 3 Ca (Na) seawater. The half time for recovery of loaded axons poisoned with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and iodoacetic acid (IAA) is about the same as control axons. Axons placed in 40 mM Na choline seawater (to reduce chemical gradient for Na) or in 40 mM Na, 410 mM K seawater to reduce the electrochemical gradient for Na to near zero either fail to lose previously loaded Ca or gain further Ca.
The Journal of General Physiology 04/1979; 73(3):327-42. · 3.84 Impact Factor
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ABSTRACT: The influx of magnesium from seawater into squid giant axons has been measured under conditions where internal solute control in the axon was maintained by dialysis. Mg influx is smallest (1 pmol/cm2 sec) when both Na and ATP have been removed from the axoplasm by dialysis. The addition of 3 mM ATP to the dialysis fluid gives a Mg influx of 2.5 pmol/cm2 sec while the addition of [Na]i and [ATP]i gives 3 pmol/cm2 sec as a value for Mg influx; this corresponds well with fluxes measured in intact squid giant axons. The Mg content of squid axons is 6 mmol/kg axoplasm; this is unaffected by soaking axons in Li or Na seawater for periods of up to 100 min.
Journal of Membrane Biology 11/1978; 43(2-3):243-50. · 1.81 Impact Factor
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FEBS Letters 11/1977; 82(2):197-200. · 3.54 Impact Factor
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ABSTRACT: Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 mumol/kg axoplasm. Axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca(Na) seawater show gains of 18 mumol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 mumol/kgxh. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca](i) is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca (choline) seawater cause increases in [Ca](i). In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na](0) (40 perecent Na) is as effective as 450 mM [Na](0) (100 percent Na) in maintaining a normal [Ca](1); lower [Na] causes an increase in [Ca](i). If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Ca](1) is not loading with high [Ca](0) or low [Na](0) solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca](1) that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca](i) in 1 Ca (Na) seawater.
The Journal of General Physiology 10/1977; 70(3):329-53. · 3.84 Impact Factor
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ABSTRACT: Changes in ionized calcium were studied in axons isolated from living squid by measuring absorbance of the Ca binding dye Arsenazo III using multiwavelength differential absorption spectroscopy. Absorption changes measured in situ were calibrated in vitro with media of ionic composition similar to axoplasm containing CaEGTA buffers. Calcium loads of 50-2,500 mumol/kg axoplasm were induced by microinjection, by stimulation in 112 mM Ca seawater, or by soaking in choline saline with 1-10 mM Ca. Over this range of calcium loading of intact axoplasm, the ionized calcium in the axoplasm rose about 0.6 nM/muM load. Similar loading in axons preteated with carbonyl cyanide 4- trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased ionized calcium by 5-7 percent of the imposed load, i.e. 93-95 percent of the calcium load was buffered by a process insensitive to FCCP. This FCCP- insensitive buffer system was not saturated by the largest calcium loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in ionized calcium which could be correlated with the extent of the load. Analysis of results indicated that, whereas only 6 percent of the endogenous calcium in fresh axons is stored in the FCCP-sensitive (presumably mitochondrial) buffer system, about 30 percent of an imposed exogenous load in the range of 50-2,500 muM is taken up by this system.
The Journal of General Physiology 10/1977; 70(3):355-84. · 3.84 Impact Factor
