A comparison of measurements of intracellular Ca by Ca electrode and optical indicators

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.     

A comparison of measurements of intracellular Ca by Ca electrode and optical indicators

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.     

Ionized calcium concentrations in squid axons

Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-mul drop of aequorin confined to a plastic dialysis tube of 140-mum diameter located axially. Ionized Ca had a mean value of 20 x 10(-9) M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasma was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10(-9) M as calibrated against CaEGTA/EGTA buffer mixtures. Wth a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca.     

The control of ionized calcium in squid axons

Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 μmol/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 μmol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 μmol/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 "late" Ca channel in squid axons

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 influence of chemical agents on the level of ionized [Ca2+] in squid axons

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.     

Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.     

Calcium content and net fluxes in squid giant axons

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.     

Intracellular calcium transients and developed tension in rat heart muscle. A mechanism for the negative interval-strength relationship

The purposes of the present study were to determine (a) whether changes of intracellular [Ca2+] (Cai) can account for the decrease of developed tension observed in rat heart muscle when stimulation rate is increased, and (b) whether the effect of stimulation rate on Cai is altered in conditions in which the rate of repriming of the sarcoplasmic reticulum (SR) is altered, as when perfusate [Ca2+] (Cao) is increased, and in heart muscle from senescent animals. The photoprotein aequorin was used to monitor Cai in rat papillary muscles. In muscles from 6-mo-old rats, increasing the stimulation rate in the range 0.2-0.66 Hz led to parallel decreases of both the aequorin light transient and developed tension when Cao was 2 mM. When Cao was increased to 4 mM, changes in the stimulation rate had less effect on both the light transient and tension. At 8 mM Cao, changing the stimulation rate had no effect on either the light transient or developed tension. Papillary muscles from 24-mo-old rats, in which SR function is likely to be depressed, exhibited a prolonged Ca2+ transient and twitch. At a Cao of 4 or 8 mM, increasing the stimulation rate from 0.33 to 0.66 Hz still led to decreases in the size of the aequorin light transient and developed tension in these muscles. Developed tension and aequorin light responded to increases of Cao in the same way in both groups of muscles. We conclude that under the conditions of our experiments, developed tension is determined by Cai. The negative interval-strength relationship observed when Cao is in the physiological range can be accounted for by a time-dependent recycling of Ca2+ by the SR. The effects of increasing Cao and the age-related differences observed at high Cao can also be accounted for using this model.     

Microtubules and actin in giant nerve fibers of the spiny lobster, Panulirus argus

Giant axons of the spiny lobster, Panulirus argus, are filled with microtubules that are decorated with fine, irregular filaments. Mitochondria and membrane-limited clear vesicles are the only other distinguishable elements in the axoplasm and are located around the periphery of the axon near the axolemma. Neither 100 A neurofilaments nor 70 A microfilaments are evident in fixed, intact axons or in negatively stained axoplasm. Actin-like microfilaments are a prominent constituent of the glial cells that closely ensheathe the axons, and gel electrophoresis studies suggest that most of the actin in the nerve fibers is located in the glia rather than in the axons. Studies of isolated axoplasm indicate that microtubules are the primary elements stabilizing the axoplasm. The microtubules in the isolated axoplasm are disrupted by Ca2+ in the medium in the presence of protease inhibitors.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

Intracellular calcium buffering capacity in isolated squid axons

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 μmol/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/μM 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 μM is taken up by this system.     

The effects of nitroprusside and putative agonists on guanylate cyclase activity in squid giant axons

cGMP content of axoplasm from the giant axon of Loligo forbesi was investigated after subjecting the axon to various treatments. Repetitive electrical stimulation or depolarisation by high K+ caused no change in cGMP content. Glutamate and serotonin were also without effect. The nicotinic agonist carbachol (100 microM) increased cGMP levels by 90% (n = 5). A large transient elevation of cGMP content was evoked by external nitroprusside (10 nM-20 microM in intact axons. Nitroprusside injected into both extruded axoplasm and intact axons also increased cGMP content, the stimulation being considerably higher in intact axons where the axolemma was also present. Nitroprusside was also active in axons where the soluble cytoplasmic components were washed out by internal perfusion.     

The incorporation of microinjected 14C-amino acids into TCA insoluble fractions of the giant axon of the squid

Giant squid axons were microinjected with serine, valine and leucine-C¹⁴ under controlled electrophysiological conditions. These amino acids are incorporated into TCA insoluble fraction in the isolated axon. This incorporation is higher in the stimulated axons as compared to non-stimulated ones. By processing separately the axoplasm and axon sheath, it was found that the last one is responsible almost entirely for the observed incorporation. Through differential centrifugation of homogenates of microinjected axons was shown that the highest incorporation occurred in the 1500 × g sediment, which probably corresponds to membranes. The incorporation of amino acids in stimulated axons, is strongly inhibited by chloramphenicol and actinomycin D.     

Mitochondria and other calcium buffers of squid axon studied in situ

Continuous nondestructive monitoring of intracellular ionized calcium in isolated squid axons by differential absorption spectroscopy (using arsenazo III and antipyrylazo III) was used to study uptake of calcium by carbonyl cyanide, p-trifluoromethoxy-phenylhydrazone (FCCP)- and (or) cyanide (CN)-sensitive and insensitive constituents of axoplasm. Known calcium loads imposed on the axon by stimulation produced proportional increments of free axoplasmic calcium. Measurement of increments in ionized calcium as a function of load confirmed earlier reports of buffering in normal and FCCP- and (or) CN-poisoned axons. Measurement of rates of calcium uptake by presumed mitochondria showed little uptake at ambient Ca below 200--400 nM, with sigmoidal rise to about 20--30 mumol/kg axoplasm per min (calculated to be about 200 mmol/kg mitochondrial protein per min) at 50 micrometer, indicating a functional threshold for presumed mitochondrial uptake well above physiological ionized calcium concentration. Treatment of stimulated axons with cyanide, to release calcium from presumed mitochondria, showed that the sensitivity to cyanide decreased progressively with time after stimulation (t 1/2 = 3--10 min) implying transfer of sequestered calcium into a less metabolically labile form.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

The effects of manganese and changes in internal calcium on Na-Ca exchange fluxes in the intact squid giant axon.

The effects of manganese chloride were studied on Na-Ca exchange fluxes from intact squid axons. Ca uptakes and Cao-dependent sodium efflux were inhibited half-maximally by 3-7 mM MnCl2. Mn inhibition appears less during Nao-Cai exchange (half-maximal inhibition; 30 mM) than that during Cao-Nai exchange, even when both fluxes were activated with 100 mM Na. The effects of changes in [Ca2+i], effected by Ca-EGTA injection or inhibition of mitochondrial Ca uptake by ruthenium red, were examined on the reverse (Cao-Nai) exchange mode. Ca-EGTA mixtures, designed to raise [Ca2+i] above 2 microM, inhibited Cao-Nai exchange fluxes. Ruthenium red inhibited mitochondrial Ca buffering to effect increases in Cai in the absence of Ca chelators; it activated Nao-Cai exchange fluxes but had little effect on Cao-Nai exchange despite similar reported Km for Cai. The results reflect the difficulty in demonstrating the stimulatory effect of [Ca2+i] on Cao-Nai exchange fluxes in intact axons.     

Magnesium efflux in dialyzed squid axons

The efflux of Mg++ from squid axons subject to internal solute control by dialysis is a function of ionized [Mg], [Na], [ATP], and [Na]o. The efflux of Mg++ from an axon with physiological concentrations of ATP, Na, and Mg inside into seawater is of the order of 2-4 pmol/cm2s but this efflux is strongly inhibited by increases in [Na]i, by decreases in [ATP]i, or by decreases in [Na]o. The efflux of Mg++ is largely independent of [Mg]i when ATP is at physiological levels, but in the absence of ATP reaches half the value of Mg efflux in be presence of ATP when [Mg]i is about 4 mM and [Na] 40 mM. Half-maximum responses to ATP occur at about 350 micronM ATP into seawater with Na either present or absent. The Mg efflux mechanism has many similarities to the Ca efflux system in squid axons especially with respect to the effects of ATP, Nao, and Na on the flux. The concentrations of free Mg and Ca in axoplasm differ, however, by a factor of 10(5) while the observed fluxes differ by a factor of 10(2).     

Sodium efflux in Myxicola giant axons

Several properties of the Na pump in giant axons from the marine annelid Myxicola infundibulum have been determined in an attempt to characterize this preparation for membrane transport studies. Both NaO and KO activated the Na pump of normal microinjected Myxicola axons. In this preparation, the KO activation was less and the NaO activation much greater than that found in the squid giant axon. However, when the intracellular ATP:ADP ratio of the Myxicola axon was elevated by injection of an extraneous phosphagen system, the K sensitivity of Na efflux increased to the magnitude characteristic of squid axons and the activating effect of NaO disappeared. Several axons were injected with Na2SO4 in order to determine the effect of elevated Nai on the Na efflux. Increasing Nai enhanced a component of Na efflux which was insensitive to ouabain and dependent on [Ca] in Na-free (Li) seawater. After subtracting the CaO-dependent fraction, Na efflux was related linearly to [Na]i in all solutions except in K-free (Li) seawater, where it appeared to reach saturation at high [Na]i.     

Incorporation of H3-uridine and the isolation and characterization of RNA from squid axon

H₃-Uridine microinjected in the giant axons of the squid is incorporated in a TCA insoluble material. There is no difference between stimulated and resting axons as to the amount incorporated. The amount incorporated is increased if the stimulation precedes the microinjection of the tracer. RNA was purified and characterized from the axoplasm, axon sheaths and from a purified membrane preparation obtained from squid retinal nerve.