Ruthenium red selectively depletes inositol 1,4,5-trisphosphate-sensitive calcium stores in permeabilized rabbit pancreatic acinar cells

Rabbit pancreatic acinar cells, permeabilized by saponin treatment, rapidly accumulated 3.5 nmol of Ca²⁺/mg protein in an energy-dependent pool when incubated at an ambient free Ca²⁺ concentration of 100 nm. Maximal loading of the internal stores was reached at 10 min and remained unchanged thereafter. Complete inhibition of the Ca²⁺ pump with thapsigargin revealed that this plateau was the result of a steady-state between slow Ca²⁺ efflux and ATP-driven Ca²⁺ uptake. Sixty percent of the pool could be released by Ins(1,4,5)P₃, whereas GTP released another twenty percent. The striking finding of this study is that the energy-dependent store could also be released by ruthenium red. Uptake experiments in the presence of ruthenium red revealed that the dye, at concentrations below 100 m, selectively reduced the size of the Ins(1,4,5)P₃-releasable pool. Ruthenium red had no effect on the half-maximal stimulatory concentration of Ins(1,4,5)P₃. At concentrations beyond 100 m, the dye also affected the GTP-releasable pool. Comparison with thapsigargin revealed that ruthenium red released Ca²⁺ from stores loaded to steady-state at a rate markedly faster than can be explained by inhibition of the ATPase alone. From the data presented, we concluded that ruthenium red selectively releases Ca²⁺ from the Ins(1,4,5)P₃-sensitive store by activating a Ca²⁺ release channel, whereas Ca²⁺ release from the GTP-sensitive store is predominantly caused by inhibition of the Ca²⁺ pump. The postulated ruthenium red-sensitive Ca²⁺ release channel might be similar to the ryanodine-receptor in muscle.The research of Dr. P.H.G.M. Willems has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences.     

Effects of quinine on Ca++-induced K+ efflux from human red blood cells

Summary The Ca⁺⁺-mediated increase in K⁺-permeability of intact red blood cells (Gardos effect) was initiated by exposing cells to known concentrations of Ca⁺⁺ (using EGTA buffers) in the presence of the ionophore A23187. The potency of quinine, an inhibitor of the response, was found to depend on the external K⁺ concentration. In K⁺-free solutions the concentration of quinine to achieve 50% inhibition (K ₅₀) was 5 m, but at 5mm K⁺ the required concentration was increased 20-fold to 100 m. An increase in internal Na⁺ had the opposite effect, allowing a high potency of quinine despite the presence of external K⁺. Alterations in the internal K⁺ level, on the other hand, were without effect on theK ₅₀, suggesting that the membrane potential is not a factor. This conclusion is supported by the lack of effect on quinine inhibition of substitution of Cl^– by NO ₃ ^– , a considerably more permeant anion. The data are consistent with the hypothesis that quinine inhibits by competitively displacing K⁺ from an external binding site, the reported K⁺-activation site for the Ca⁺⁺-mediated K⁺-permeability.     

The interrelationships between sodium ion,calcium transport and oxygen utilization in the isolated chick chorioallantoic membrane

Summary The interrelationships between sodium ion, calcium transport and oxygen utilization have been investigated in the chick chorioallantoic membrane. The oxygen uptakes of the two surface layers of the tissue, the ectoderm and the endoderm, were separated into their basal, Na⁺ dependent and Ca⁺⁺ dependent components. The endoderm has a basal rate of respiration of 3.6 liters O₂/cm²/hr and a Na⁺ dependent component of 1.4 liters O₂/cm²/hr. The ectoderm has a basal rate of respiration of about 3.5 liters O₂/cm²/hr, and Na⁺ and Ca⁺⁺ dependent components of 1.1 and 3.6 liters O₂/cm²/hr, respectively. The rate of ectodermal calcium transport and calcium-stimulated oxygen uptake is strictly dependent on the presence of sodium in the bathing medium, and complex kinetics are observed as a function of sodium concentration. On the other hand, in 140mm Na⁺ the rate of calcium transport exhibits simple saturation kinetics as a function of calcium concentration. Ca⁺⁺/O₂ ratios determined for many different rates of transport give a ratio of about 0.5, a value much lower than similar ratios determined for other transport mechanisms. The calcium transport mechanism in the ectoderm responds to changes in transport rate very sluggishly, taking 30 to 50 min to give a maximum response. The differences between the calcium transport mechanism in this membrane and other known transport systems are discussed and it is suggested that these differences may represent the adaptations necessary for transcellular calcium transport.     

A minimum mechanism for Na+−Ca++ exchange: Net and unidirectional Ca++ fluxes as functions of ion composition and membrane potential

Summary Both simultaneous and consecutive mechanisms for Na⁺–Ca⁺⁺ exchange are formulated and the associated systems of steady-state equations are solved numerically, and the net and unidirectional Ca⁺⁺ fluxes computed for a variety of ionic and electrical boundary conditions. A simultaneous mechanism is shown to be consistent with a broad range of experimental data from the squid giant axon, cardiac muscle and isolated sarcolemmal vesicles. In this mechanism, random binding of three Na⁺ ions and one Ca⁺⁺ on apposing sides of a membrane are required before a conformational change can occur, translocating the binding sites to the opposite sides of the membranes. A similar (return) translocation step is also permitted if all the sites are empty. None of the other states of binding can undergo such translocating conformational changes. The resulting reaction scheme has 22 reaction steps involving 16 ion-binding intermediates. The voltage dependence of the equilibrium constant for the overall reaction, required by the 31 Na⁺Ca⁺⁺ stoichiometry was obtained by multiplying and dividing, respectively, the forward and reverse rate constants of one of the translocational steps by exp(–FV/2RT). With reasonable values for the membrane density of the enzyme (120 sites m²) and an upper limit for the rate constants of both translocational steps of 10⁵·sec^–1, satisfactory behavior was obtainable with identical binding constants for Ca⁺⁺ on the two sides of the membrane (10⁶ m ^–1), similar symmetry also being assumed for the Na⁺ binding constant (12 to 60m ^–1). Introduction of order into the ion-binding process eliminates behavior that is consistent with experimental findings.     

Lysozyme acts as a chemorepellent and secretagogue in Paramecium by activating a novel receptor-operated Ca++ conductance

Using combined intracellular recordings and behavioral bioassays, it was found that lysozyme has two different effects in Paramecium, depending upon the concentrations used. At low concentrations (0.5 nm to 1.0 m) it acts as an effective chemorepellent that causes reliable electrophysiological changes. Lysozyme-induced somatic depolarizations, isolated by blocking K⁺ channels with Cs-TEA, showed concentration dependencies that were well correlated with chemorepulsion. Ion dependency experiments showed that these were Ca⁺⁺ based depolarizations. Addition of either Na⁺ or Mg⁺⁺ improves chemorepulsion by providing additional depolarizations. Both the depolarizations and chemorepulsion were blocked by 10 m neomycin, suggesting that the depolarization is necessary for this chemosensory transduction event. At higher concentrations (100 m), lysozyme is a secretagogue. A transient inward current, recorded in Ca⁺⁺ alone solutions with Cs-TEA present, was seen in response to high lysozyme concentrations. The amplitude of this inward current was well correlated with exocytosis. Addition of neomycin (1.0 mm) eliminated both the inward current and exocytosis, suggesting a causal relationship. Neither amiloride or W-7, compounds previously suggested to affect the electrophysiological responses to secretagogues, had any significant effects. The mucopolysaccharide hydrolysis activity of lysozyme was not required for any of these responses. We propose that Paramecium have a high affinity receptor on the body plasma membrane that responds to either lysozyme or a related compound to cause an increase in a novel body Ca⁺⁺ conductance. This receptor-operated Ca⁺⁺ conductance causes membrane depolarization and chemorepulsion at low concentrations and triggers a sufficient Ca⁺⁺ influx at high concentrations to cause exocytosis.We thank Drs. C. Kung and R. Preston for sharing mutants and Drs. H. Machemer, A. Turkewitz and K. Clark for their comments on the first draft of this work. This was supported by NSF grants BNS8916228 and MCB9410756 to TMH and a grant from the American Diabetes Association to BHS.     

Role of magnesium in the (Ca2++Mg2+)-stimulated membrane ATPase of human red blood cells

Summary (Ca²⁺+Mg²⁺)-stimulated ATPase of human red cell membranes as a function of ATP concentration was measured at fixed Ca²⁺ concentration and at two different but constant Mg²⁺ concentrations. Under the assumption that free ATP rather than Mg-ATP is the substrate, a value forK _m (for ATP) of 1–2m is found which is in good agreement with the value obtained in the phosphorylation reaction by A.F. Rega and P.J. Garrahan (1975.J. Membrane Biol. 22:313). Mg²⁺ increases both the maximal rate and the affinity for ATP, whereas Ca²⁺ increases the maximal rate without affectingK _m for ATP.As a by-product of these experiments, it was shown that after thorough removal of intracellular proteins the adenylate kinase reaction at approximately 1mm substrate concentration is several times faster than maximal rate of (Ca²⁺+Mg²⁺)-ATPase in red cell membranes.     

Calcium entry in rabbit corneal epithelial cells: Evidence for a nonvoltage dependent pathway

We performed experiments to elucidate the calcium influx pathways in freshly dispersed rabbit corneal epithelial cells. Three possible pathways were considered: voltage-gated Ca⁺⁺ channels, Na⁺/Ca⁺⁺ exchange, and nonvoltage-dependent Ca⁺⁺-permeable channels. Whole cell inward currents carrying either Ca⁺⁺ or Ba⁺⁺ were not detected using voltage clamp techniques. We also used imaging technology and the Ca⁺⁺-sensitive ratiometric dye fura 2 to measure changes in intracellular Ca⁺⁺ concentration ([Ca]_i). Bath perfusion with NaCl Ringer's solution containing the calcium channel agonist Bay-K-8644 (1 m), or Ni⁺⁺ (40 m), a blocker of many voltage-dependent calcium channels, did not affect [Ca⁺⁺]_i. Membrane depolarization with a KCl Ringer's bath solution resulted in a decrease in [Ca⁺⁺]_i. These results are inconsistent with the presence of voltage gated Ca⁺⁺ channels. Nonvoltage gated Ca⁺⁺ entry, on the other hand, would be reduced by membrane depolarization and enhanced by membrane hyperpolarization. Agents which hyperpolarize via stimulation of K⁺ current, such as flufenamic acid, resulted in an increase in ratio intensity. The cells were found to be permeable to Mn⁺⁺ and bath perfusion with 5 mm Ni⁺⁺ decreased [Ca⁺⁺]_i suggesting that the Ca⁺⁺ conductance was blocked. These results are most consistent with a nonvoltage gated Ca⁺⁺ influx pathway. Finally, replacing extracellular Na⁺ with Li⁺ resulted in an increase in [Ca⁺⁺]_i if the cells were first Na⁺-loaded using the Na⁺ ionophore monensin and ouabain, a Na⁺-K⁺-ATPase inhibitor. These results suggest that Na⁺/Ca⁺⁺ exchange may also regulate [Ca⁺⁺] in this cell type.The authors are grateful to Chris Bartling for expert technical assistance with the imaging experiments, Helen Hendrickson for cell preparation, and Jonathon Monck for helpful discussions regarding imaging technology. This work was supported by National Institutes of Health grants EYO3282, EYO6005, DK08677, and an unrestricted award from Research to Prevent Blindness.     

Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin

We have studied the activities of Ca²⁺-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca²⁺-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg²⁺ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca²⁺-stimulated ATPase without affecting its sensitivity to Ca²⁺ or Mg²⁺ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca²⁺-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg²⁺ATP. DTT increased the affinity of the enzyme to Mg²⁺ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca²⁺-stimulated ATPase to Ca²⁺. DTT protected the Ca²⁺-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca²⁺-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca²⁺-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca²⁺-stimulated ATPase activity in sarcolemmal preparations.     

A thermodynamic analysis of the correlation between active Na+ transport and the rate of oxygen consumption in epithelia

Summary Insulin decreased markedly the adenylyl cyclase activity associated with fat cell membranes purified by centrifugation in sucrose gradients. The hormone effect was not readily evident in crude membrane preparations.The kinetics of this effect indicate that some time was required for the onset of the insulin-induced inactivation. This lag period decreased when the insulin concentration was increased. The hormone dose dependence for adenylyl cyclase inactivation measured at a fixed time (3 min) showed a 10 to 15% decrease in activity at 1 to 30 U per ml insulin; 30 to 40% at 100 to 1000 U per ml; and 75% at 0.1 U per ml.The insulin effect was completely abolished by 0.1mm GMP-P(NH)P, 10mm fluoride, or 50 ng per ml glucagon, or by increasing the Mn⁺⁺ concentration to 4mm. In addition, it was partially reversed by the addition of a fraction from the sucrose gradient, which contained soluble factors.The kinetics of the adenylyl cyclase-catalyzed reaction were studied using ATP or AMP-P(NH)P as adenylyl donor, and Mn⁺⁺ or Mg⁺⁺ as divalent cation, in the absence or presence of insulin. With ATP and Mg⁺⁺ there was a striking reduction of the transient reaction rates after 1.5 min of incubation. Under these conditions the insulin effect was not evident. On the contrary, with ATP and Mn⁺⁺ this spontaneous reduction of activity was less evident; however, in the presence of insulin there was a clear and marked reduction of the transient reaction rate measured after 1.5 min of incubation. With AMP-P(NH)P the kinetic data were qualitatively similar to those observed with ATP.It is concluded that under certain assay conditions adenylyl cyclase may be converted to an inactive enzyme form, and that such a conversion is more evident in the presence of Mg⁺⁺ than with Mn⁺⁺. In the latter case, insulin appears to enhance the rate of this conversion.     

Characterization of fructose-1,6-diphosphate-insensitive catabolic glycerol kinase ofPseudomonas aeruginosa

Glycerol kinase is induced in cells ofPseudomonas aeruginosa strain PAO when grown in the presence of glycerol or glycerol-3-phosphate. The enzyme was isolated from the soluble cytoplasmic fraction of cell extracts and purified 500-fold by ammonium sulfate precipitation and chromatography on columns of Sephadex G-25, DEAE-Sephadex, hydroxyapatite, and Sephadex G-200. A molecular weight of 120,000 was estimated by gel filtration of the catalytically active enzyme. In polyacrylamide gel electrophoresis the purified product contained one major band of Coomassie Blue staining material. The enzyme exhibited an apparent Km of 40 M for glycerol and 23 M for ATP. Of the nucleotide triphosphates tested, only ATP served as a phosphoryl group donor. Mg⁺⁺ or Mn⁺⁺ was required for activity, although a threefold greater concentration of Mn⁺⁺ was required when Mn⁺⁺ substituted for Mg⁺⁺. In contrast to most other catabolic glycerol kinases in bacteria, the enzyme was not inhibited by fructose-1,6-diphosphate nor by other tested metabolites.     

Effect of culture medium ions on chromate reduction by resting cells of Agrobacterium radiobacter

The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe²⁺ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 m Mg²⁺, 20 m Ca²⁺ and 3.6 m Mn²⁺. Chromate reduction was not affected by the addition of MgCl₂, CdCl₂, ZnCl₂, MnCl₂, Na₂SO₄ (1000 m), and Na₂MoO₄ (100 m) to the activity assays. However, activity was inhibited by the presence of Na₂SO₄ (10 mm), Na₂MoO₄ (200 m) and ferric citrate.     

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Summary A vanadate-sensitive H⁺-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H⁺-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H⁺ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H⁺ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na₃VO₄) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK _i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na₃VO₄). ATPase activity of the reconstituted vesicles showed aK _i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.     

Rundown and reactivation of ATP-sensitive potassium channels (KATP) in mouse skeletal muscle

Dissociated single fibers from the mouse flexor digitorum brevis (FDB) muscle were used in patch clamp experiments to investigate the mechanisms of activation and inactivation of K_ATP in mammalian skeletal muscle. Spontaneous rundown of channel activity, in many excised patches, occurred gradually over a period of 10–20 min. Application of 1.0 mm free-Ca²⁺ to the cytoplasmic side of the patch caused irreversible inactivation of K_ATP within 15 sec. Ca²⁺-induced rundown was not prevented by the presence of 1.0 m okadaic acid or 2.0 mg ml^– of an inhibitor of calcium-activated neutral proteases, a result consistent with the conclusion that phosphatases or calcium-activated neutral proteases were not involved in the rundown process. Application of 1.0 mm Mg.ATP to Ca²⁺inactivated K_ATP caused inhibition of residual activity but little or no reactivation of the channels upon washout of ATP, even in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase (10 U ml^–1). Mg.ATP also failed to reactivate K_ATP, even after only partial spontaneous rundown, despite the presence of channels that could be activated by the potassium channel opener BRL 38227. Nucleotide diphosphates (500 m; CDP, UDP, GDP and IDP) caused immediate and reversible opening of Ca²⁺-inactivated K_ATP. Reactivation of K_ATP by ADP (100 m) increased further upon removal of the nucleotide. In contrast to K_ATP from cardiac and pancreatic cells, there was no evidence for phosphorylation of K_ATP from the surface sarcolemma of dissociated single fibers from mouse skeletal muscle. The small degree of activation occasionally observed following application of 10 m or 1.0 mm Mg.ATP could have been due to the generation of ADP from ATP hydrolysis and not through phosphorylation. Data are consistent with the suggestion that Ca²⁺ inactivation of K_ATP involves a gating mechanism that can be reopened by nucleotide diphosphates.M.H. is supported by the Medical Research Council.     

Characteristics of the ATP-ase activity of isolated neurons of rabbit

Summary Isolated and homogenised Deiters' neurons from the lateral vestibular nucleus of rabbit in a Krebs-Ringer solution containing no added Mg⁺⁺, 1.3 moles/ ml and 5 moles/ml Mg⁺⁺, broke down ATP at the maximal rate of 0.29+-0.20, 2.40+–0.20, and 5.95+–0.63 moles/cell/hr. In 1.3 mM Mg⁺⁺ solution the single cell homogenates took up phosphate at the mean rate of 2.6+–0.2 moles/cell/hr. If the rabbits were injected 1 hour before with 20 mg/kg body weight of 2-amino-1-propene-1,1,3, tricarbonitrile (triap), the breakdown of ATP in these latter media was 0.82+–0.44, 2,5+–0.60, and 6.7+– 1.1 moles/cell/hr, respectively, and the quantity of inorganic liberated did not decrease.     

Ammonium photoproduction by free and immobilized cells of Chlamydomonas reinhardtii

Summary Free-living or immobilized Chlamydomonas reinhardtii cells photoproduce ammonium from nitrite in a medium containing 1 mM of l-methionine-d,l-sulphoximine (MSX). Ammonium is accumulated in the medium to 8 mM final concentration, which inhibits nitrite uptake by the MSX-treated cells and consequently the excretion of ammonium is blocked. However, if ammonium was removed from the medium and nitrite and MSX periodically restored, the photoproduction process could be maintained over 96 h, with a final ammonium concentration of about 18 mM for free-living cells and 28 mM for immobilized ones. The MSX-treated cells showed a photoproduction productivity of 1300 mol NH ₄ ⁺ · mg chlorophyll (Chl)^-1, with an average production rate of 14 mol NH ₄ ⁺ · mg Chl^-1 per hour, for calcium alginate-entrapped cells, while the corresponding data for free-living ones was 650 mol NH ₄ ⁺ · mg Chl^-1 and 6.7 mol NH ₄ ⁺ · mg Chl^-1 per hour, respectively. Immobilized cells showed a significant increase in the nitrite uptake rate, probably due to a change in membrane permeability as a consequence of cell-matrix interactions.     

Adenosine discriminates between the caffeine and adenine nucleotide sites on the sheep cardiac sarcoplasmic reticulum calcium-release channel

Calcium-release channels of sheep cardiac sarcoplasmic reticulum were incorporated into phosphatidylethanolamine bilayers and single channel currents were recorded under voltage-clamp conditions. The effect of adenosine on single channel conductance and gating was investigated, as were the interactions between adenosine and caffeine and adenosine and ,-methylene ATP.Addition of adenosine (0.5–5 mm) to the cytosolic but not the luminal side of the membrane increased the open probability of single calcium-activated calcium-release channels by increasing the frequency and duration of open events, yielding an EC₅₀ of 0.75 mm at 10 m activating Ca²⁺.Addition of 1 mm caffeine potentiated the effects of adenosine at 10 or 100 m-activating cytosolic calcium, but had no effect on the inability of adenosine to activate the channel at 80 pmcalcium, suggesting discrete sites of action on the calcium-release channel for adenosine and caffeine. In contrast, addition of 100 m ,-methylene-ATP decreased single channel open probability in the presence of adenosine, suggesting that these compounds act on the same site on the channel.Activation of single channel opening by adenosine, or by adenosine together with caffeine, had no effect on single channel conductance or the Ca²⁺/Tris⁺ permeability ratio. Channels activated by adenosine were characteristically modified by ryanodine and blocked by m ruthenium red or mm magnesium.These results show that adenosine activates the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel by increasing the frequency and duration of open events in a Ca²⁺-dependent manner. The receptor site on the channel for adenosine is distinct from that for caffeine but probably the same as that for adenine nucleotides.This work was supported by the British Heart Foundation.     

Functional heterogeneity of the sarcoplasmic reticulum within sarcomeres of skinned muscle fibers

Summary Precipitation of Ca oxalate in the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers caused an increase in light scattering which was proportional to the amount of Ca accumulated per unit fiber volume. The increase in scattering was used to measure net accumulation rates and steady-state Ca capacities of the sarcoplasmic reticulum in single fibers. The data obtained were qualitatively and quantitatively similar to those reported for isolated vesicle preparations.Under conditions in which Ca was not depleted from the medium, Ca accumulation was linear with time over much of its course. Steady-state capacities were independent of the Ca concentration; uptake rates were half-maximal at 0.5 m Ca⁺⁺ and saturated above about 1.0 m. Both rate and capacity varied with the oxalate concentration, being maximal at oxalate concentrations >=5mm and decreasing in proportion to one another at lower concentrations, with a threshold near 0.25mm. At the lower loads, electron micrographs showed many sarcoplasmic reticulum elements empty of precipitate alongside others that were full, whereas virtually all were filled in maximally loaded fibers. These data indicate that the Ca oxalate capacity of each fiber varies with the number and volume of elements in which Ca oxalate crystals can form at a given oxalate concentration, and that individual regions of the sarcoplasmic reticulum within each sarcomere differ in their ability to support Ca oxalate precipitation. Our working hypothesis is that this range in ability to form Ca oxalate crystals involves differences in ability to accumulate and retain ionized Ca inside the sarcoplasmic reticulum.     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

Apparent loss of calcium-activated potassium current in internally perfused snail neurons is due to accumulation of free intracellular calcium

Summary Internal perfusion ofHelix neurons with a solution containing potassium aspartate, MgCl₂, ATP, and HEPES causes the calcium-activated potassium current (I _K(Ca)) evoked by depolarizing voltage steps to decrease with time. When internal free Ca⁺⁺ is strongly buffered to 10^–7 m by including 0.5mm EGTA and 0.225mm CaCl₂ in the internal solution,I _K(Ca) remains constant for up to 3 hours of perfusion. In cells whereI _K(Ca) is small at the start of perfusion, perfusion with the strongly buffered 10^–7 m free Ca⁺⁺ solution produces increases inI _K(Ca) which ultimately saturate. In cells perfused with solutions buffered to 10^–6 m free Ca⁺⁺,I _K(Ca) is low and does not change with perfusion. These results lead us to conclude thatI _K(Ca) is stable in perfusedHelix neurons and that the apparent loss ofI _K(Ca) seen initially with perfusion is due to accumulation of cytoplasmic calcium. Since the calcium current (I _Ca) provides the Ca⁺⁺ which activatesI _K(Ca) during a depolarizing pulse,I _Ca is also stable in perfused cells when free intracellular Ca⁺⁺ is buffered.Perfusion with 1 m calmodulin (CaM) produces no effect onI _K(Ca) with either 10^–7 or 10^–6 m free internal calcium. Inhibiting endogenous CaM by including 50 m trifluoperazine (TFP) in both the bath and the internal perfusion solution also produces no effect onI _K(Ca) with 10^–7 m free internal calciu. It is concluded that CaM plays no role inI _K(Ca) activation.     

Selective effects of cyanide (100 μM) on the excitatory amino acid-induced elevation of intracellular calcium levels in neuronal culture

The effect of low concentrations of cyanide on the excitatory amino acid-induced elevations of intracellular calcium levels ([Ca²⁺]_i) was studied in cerebellar granule cells using ratio fluorometry with fura-2. Glutamate, kainate, N-methyl-d-aspartate (NMDA), quisqualate (50 M, each) and membrane depolarization by 40 mM KCl caused elevations of [Ca²⁺]_i which were 10-, 10-, 3-, 2.3-, 10-fold over baseline levels, respectively. Cyanide, 100 M, greatly augmented the increases in [Ca²⁺]_i induced by glutamate, kainate and NMDA but not those induced by quisqualate or KCl. In the absence of these excitatory amino acids, cyanide had no significant effect in concentrations up to 400 M. Elevations of [Ca²⁺]_i induced by quisqualate and KCl were not significantly augmented by higher concentrations of cyanide (400 M). Selective antagonists could block the effect of cyanide+the respective agonist; however, the calcium channel blockers, lanthanum and diltiazem lowered both NMDA- and kainate-induced elevations of [Ca²⁺]_i, yet neither blocked increases in calcium when 100 M cyanide was added. Collectively, these data support an interaction of cyanide with the excitatory amino acid receptor.