Repolarization of striated muscle fibers by the antifatigue agent,K-Mg-aspartate

Resting membrane potentials of isolated frog sartorius muscles were measured under a variety of conditions using intracellular glass microelectrodes. Muscle cells depolarized by the addition of 5.0 or 10.0 mM KCl to the bathing Ringer solution can be repolarized some 5 to 10 mV by the substitution of an equivalent amount of K-aspartate for KCl in the presence of 2.0 mM Mg⁺⁺. The repolarization produced by this method persists when the muscle is again placed in the initial KCl solution, thus eliminating the possibility that the hyperpolarization is due to the reduction of chloride in the bathing medium. If for some reason the resting membrane potential of the muscle fibers is considerably below (less negative than) the normal level of 92 mV reported for muscles bathed in 2.5 mM Ringer solution, the substitution of 2.5 mM K-aspartate for the 2.5 mM KCl and the addition of 2.0 mM Mg-aspartate to the Ringer solution will, within 15 minutes, repolarize the fiber to the normal level. Magnesium ions alone will not produce the observed repolarization nor can it be attributed to a reduction in the activity of the potassium in the Ringer solution.     

Activation of Maxi-K Channels by Parathyroid Hormone and Prostaglandin E2 in Human Osteoblast Bone Cells

Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mm Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nm parathyroid hormone (PTH), 5 nm prostaglandin E₂ (PGE₂) or 0.1 mm dibutyryl cAMP + 1 μm forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mm K) the channel has a conductance of 246 ± 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (P_K/P_Na) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (P _o ) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the P _o vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mm ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE₂ or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (⁴⁵Ca⁺⁺) by MG-63 cells was stimulated in the presence of PTH and PGE₂, an effect inhibited by Nitrendipine (10 μm). Second, whereas PGE₂ stimulated the calcium-activated maxi-K channel in 2 mm Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE₂ did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mm Ca Ringer and 10 μm Nitrendipine in CAP configuration, PGE₂ (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE₂ or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx. Received: 17 September 1995/Revised: 7 December 1995     

The Roles of Calcium in Excitation-Contraction Coupling in Various Muscles of the Frog, Mouse, and Barnacle

In rectus abdominis muscles of the frog the active shorteningprovoked by 15–40 mM K was supported by Ca, Sr, and Ba,but not by Ni, Co, Mn, Cd, or Zn ions. Addition of the lattercations to a solution containing Ca decreased the responsesin a manner suggesting competitive inhibition. The shorteningof the rectus muscle found in divalent cation-free, low K solutionsis abolished by Ni, Co, Mn, and Mg. In rat muscles a transientincrease in the contractural responses to elevated potassiumwas observed when Ni was applied following partial washout ofCa. In single muscle fibers of the barnacle, development oftension was supported by Ca and Sr, and the other divalent cationswere without effect. Retention of Ca⁴⁵ in barnacle resting musclefibers soaked in solutions containing 10 mM Ca for 2 min andsubsequently washed for 10 min was 60 ± 3.1 mµMCa/g, whereas retention of Ca⁴⁵ in contracting muscles similarlyexposed to Ca⁴⁵ was 156 + 17 mµM Ca/g of fresh muscle.The results are compatible with the idea that activation ofcontraction in some types of muscles is due to entry of extracellularCa.     

Quinine and caffeine effects on 45Ca movements in frog sartorius muscle

1 mM caffeine, which produces only twitch potentiation and not contracture in frog sartorius muscle, increases both the uptake and release of ⁴⁵Ca in this muscle by about 50 %, thus acting like higher, contracture-producing concentrations but less intensely. Quinine increases the rate of release of ⁴⁵Ca from frog sartorius but not from the Achilles tendon. The thresholds for the quinine effect on ⁴⁵Ca release and contracture tension are about 0.1 and 0.5 mM, respectively, at pH 7.1. Quinine (2 mM) also doubles the uptake of ⁴⁵Ca by normally polarized muscle. However, there are variable effects of quinine upon ⁴⁵Ca uptake in potassium-depolarized muscle. Quinine (2 mM), increases the Ca, Na, and water content of muscle while decreasing the K content. Both caffeine (1 mM) and quinine (2 mM) act to release ⁴⁵Ca from muscles that have been washed in Ringer''s solution from which Ca was omitted and to which EDTA (5 mM) was added. These results, correlated with those of others, indicate that a basic effect of caffeine and quinine on muscle is to directly release activator Ca²⁺ from the sarcoplasmic reticulum in proportion to the drug concentration. The drugs may also enhance the depolarization-induced Ca release caused by extra K⁺ or an action potential. In respect to the myoplasmic Ca²⁺ released by direct action of the drugs, a relatively high concentration is required to activate even only threshold contracture, but a much lower concentration, added to that released during excitation-contraction coupling, is associated with the condition causing considerable twitch potentiation.     

Observations on “Detubulated” Muscle Fibres

GLYCEROL treatment of striated muscle^1–3 has been widely assumed to be a means of disconnecting the transverse tubular system from the surface membrane. Exposure of a sartorius muscle to Ringer plus 400 mM glycerol followed by a return to Ringer, reduces the membrane capacity of superficial fibres and the muscle is mechanically inactive even though the fibres can transmit action potentials; both these effects might be expected if the transverse tubular system was destroyed or disconnected from the extracellular fluid. We have studied this mechanical decoupling on isolated muscle fibres from the semitendinosus, examining the effects of glycerol concentration. Our results suggest that decoupling may be a complex process and in particular they show that under some circumstances both the depolarization and the loss of the twitch can be reversed by re-exposure to glycerol.     

The Role of Calcium Ions in Modulation of Impulse Activity of the Isolated Muscle Spindle of the Frog Rana temporaria

The effect of various concentrations of Ca⁺² and Mg⁺² as well as of calcium channel blockers verapamil and nifedipin on impulse activity of frog isolated muscle spindles was studied. Removal of Ca ions from the external Ringer solution was established to increase spontaneous and evoked activity of the muscle spindle. A 4- and 8-fold increase of Ca⁺² concentration produces inhibition and complete cessation of the spontaneous and evoked activity in the muscle spindle. Replacement of Ca⁺² by Mg⁺² is observed to cause no statistically significant change of the spontaneous activity of the isolated muscle spindle; at the same time, at the dynamic spindle extension, the impulse activity rate at the dynamic and static phases of the response rises. Nifedipin and verapamil, blockers of Ca⁺² channels, suppress impulse activity both in norm and on the background of increased impulse activity evoked by removal of Ca⁺² from the external solution. An increase of muscle spindle impulse activity after the removal of Ca⁺² from the external solution is accounted for by transformation of calcium channels of the muscle spindle sensory endings into selective sodium channels.     

Calcium efflux from frog twich muscle fibers

An apparatus is described which collects the effluent from the center 0.7 cm of a single muscle fiber or bundle of muscle fibers. It was used to study the efflux of ⁴⁵Ca from twitch muscle fibers. The efflux can be described by three time constants 18 ± 2 min, 300 ± 40 min, and 882 ± 172 min. These kinetics have been interpreted as those of a three-compartment system. The fastest is thought to be on the surface membrane of the muscle and of the T system. It contains 0.07 ± 0.03 mM Ca/liter of fiber and the Ca efflux is 0.11 ± 0.04 pM Ca/cm². sec. The intermediate rate compartment is thought to represent the Ca in the longitudinal reticulum. It contains approximately 0.77 mM Ca/liter. Only the efflux from this compartment increases during stimulation. The most slowly exchanging compartment is poorly defined. Neither Ca-free nor Ni-Ringer solutions alter the rate of loss from the fastest exchanging compartment. Ni apparently alters the rate of loss from the slowest compartment.     

The Initiation of Spike Potential in Barnacle Muscle Fibers under Low Intracellular Ca++

Electrical properties of the muscle fiber membrane were studied in the barnacle, Balanus nubilus Darw. by using intracellular electrode techniques. A depolarization of the membrane does not usually produce an all-or-none spike potential in the normal muscle fiber even though a mechanical response is elicited. The intracellular injection of Ca⁺⁺-binding agents (K₂SO₄ and K salt of EDTA solution, K₃ citrate solution, etc.) renders the fiber capable of initiating all-or-none spikes. The overshoot of such a spike potential increases with increasing external Ca concentration, the increment for a tenfold increase in Ca concentration being about 29 mv. The threshold membrane potential for the spike and also for the K conductance increase shifts to more positive membrane potentials with increasing [Ca⁺⁺]_out. The removal of Na ions from the external medium does not change the configuration of the spike potential. In the absence of Ca⁺⁺ in the external medium, the spike potential is restored by Ba⁺⁺ and Sr⁺⁺ but not by Mg⁺⁺. The overshoot of the spike potential increases with increasing [Ba⁺⁺]_out or [Sr⁺⁺]_out. The Ca influx through the membrane of the fiber treated with K₂SO₄ and EDTA was examined with Ca⁴⁵. The influx was 14 pmol per sec. per cm² for the resting membrane and 35 to 85 pmol per cm² for one spike. From these results it is concluded that the spike potential of the barnacle muscle fiber results from the permeability increase of the membrane to Ca⁺⁺ (Ba⁺⁺ or Sr⁺⁺).     

Comparative Phloem mobility of nickel in nonsenescent plants

⁶³Ni was applied to nonsenescent source leaves and found to be transported to sink tissues in pea (Pisum sativum L.) and geranium plants (Pelargonium zonale L.). The comparative mobilities (percent tracer transported out of source leaf ÷% ⁸⁶Rb transported) for ⁶³Ni in peas was 2.12 and in geranium 0.25. The value for the phloem mobile ⁸⁶Rb was 1.00. By contrast, the comparative mobility of ⁴⁵Ca, which is relatively immobile in the phloem, was low (0.05 in peas, 0.00 in geranium). Interruption of the phloem pathway between source and sink leaves by steam girdling almost completely inhibited ⁶³Ni accumulation in the sink leaves of both species. We conclude that Ni is transported from nonsenescent source leaves to sink tissues via the phloem of leguminous and nonleguminous plants.     

On the Inhibition of Muscle Contraction Caused by Exposure to Hypertonic Solutions

The evidence supporting a site of inhibition of excitation contraction (E-C) coupling near the plasma membrane (the "glycerol effect," the K⁺-potentiating effect) for muscle in hypertonic solution was reinvestigated. It was found, using whole frog sartorii, that there was a rehydration of muscle soaked in glycerol Ringer after 30 min and a large swelling (to 140% after 1 hr soaking) upon return of the muscle to normal Ringer, suggesting that significant amounts of glycerol enter the fibers during this time. While contrary to the original report of the glycerol effect, this finding was consistent with other studies involving the use of single fibers. Also reexamined was the potentiating effect of K⁺ on the hypertonic inhibition of muscle contraction. It was found that muscles exposed to this KCl pretreatment swell so that they are less dehydrated in hypertonic solutions, thus accounting for the observed potentiation. After being treated instead with a K₂-tartrate Ringer solution, muscles did not swell and, as determined with twitch recordings, did not display any potentiation in hypertonic solutions—even though the [K₊] was higher than an osmotically equivalent KCl solution. The evidence was thus consistent with alternative hypotheses in which inhibition of contraction occurs at a later stage in E-C coupling or involves the contractile process itself.     

Effect of external sodium and calcium on calcium efflux in frog striated muscle

Summary The effect of media with different ionic composition on calcium efflux from the dorsal head of semitendinosus muscles ofRana pipiens was studied. The reduction in the fractional loss of⁴⁵Ca, when going from normal Ringer's solution to an ONa–OCa medium, was 60%. Withdrawal of only Na or Ca from the external medium also caused a significant drop in the fractional loss (33% and 34%, respectively). The effect of different concentrations of Ca (studied in the absence of the external Na) was also studied. It was found that a linear function could describe the relationship between the calcium-dependent calcium efflux and the external calcium concentration. These results indicate that calcium efflux from frog muscle fibers consists of three major components: one that is dependent on the presence of calcium in the external medium, one that is dependent on the presence of sodium in the external medium, and one that persists in the absence of these two cations.     

Acidosis and Ca2+ distribution in myocardial tissue of flounder and rat

Summary The effect of acidosis on the myocardial Ca²⁺ distribution was examined at 15°C in ventricular strips of the flounder (Platichthys flesus) and at 30°C in atrial strips of the rat (Rattus norvegicus).Lowering the Ringer pH from 7.6 to 6.9 by increasing its CO₂ (flounder 2% to 12%, rat 4% to 14%), resulted in an elevated Ca²⁺ efflux in resting strips as well as in strips stimulated (12/min) to contraction. A decrease in pH of the Ringer used for the flounder myocardium by a lowering of bicarbonate (30 mM to 5 mM) also resulted in an elevation of the Ca²⁺ efflux, but the effect was smaller than that produced by an increased CO₂.With 11 mM Ca²⁺ and 10 mM EGTA added to the Ringer to reduce the amount of⁴⁵Ca²⁺ bound to extracellular sites, an increased CO₂ with a concomitant drop in Ringer pH resulted in an increased Ca²⁺ efflux in both myocardia. The Ca²⁺ efflux was only marginally elevated in the flounder myocardium and unchanged in that of rat when the same drop in Ringer pH was produced with a lowering in bicarbonate.In a nominally Ca²⁺-free Ringer with 0.1 mM EGTA the⁴⁵Ca²⁺ efflux was stimulated for both myocardia by an increase in CO₂.The flounder myocardium was exposed to high CO₂ in a nominally Na⁺, Ca²⁺-free Ringer and again the⁴⁵Ca²⁺ efflux increased. After a return to Na, Ca and low CO₂ in the Ringer, a higher efflux persisted in the strips being subjected to a high CO₂ than in the controls.The Ca²⁺ uptake rate was about the same at high and low CO₂ for both myocardia.Based on these results the measured increase in Ca efflux following an increase in CO₂ or a decrease in bicarbonate probably results from an elevated cytoplasmatic Ca²⁺ activity. It seems unlikely that an increased uptake rate of Ca²⁺ or a direct stimulation of Ca²⁺ transporting mechanisms in the cell membrane are responsible for the change.     

The Roles of Sodium and Potassium Ions in the Generation of the Electro-Olfactogram

In order to clarify whether or not the electronegative olfactory mucosal potentials (EOG) are generator potentials, the effects of changed ionic enviroment were studied. The EOG decreased in amplitude and in some cases nearly or completely disappeared, when Na⁺ in the bathing Ringer solution was replaced by sucrose, Li⁺, choline⁺, tetraethylammonium⁺ (TEA), or hydrazine. In the K⁺-free Ringer solution, the negative EOG's initially increased and then decreased in amplitude. In Ringer's solution with increased K⁺, the negative EOG's increased in amplitude. When K⁺ was increased in exchange for Na⁺ in Ringer's solution, the negative EOG's decreased, disappeared, and then reversed their polarity (Fig. 6). Next, when the K⁺ was replaced by equimolar sucrose, Li⁺, choline⁺, TEA⁺, hydrazine, or Na⁺, the reversed potentials recovered completely only in Na⁺-Ringer's solution, but never in the other solutions. Thus, the essential role of Na⁺ and K⁺ in the negative EOG's was demonstrated. Ba⁺⁺ was found to depress selectively the electropositive EOG, but it hardly decreased and never increased the negative EOG. Hence, it is concluded that Ba⁺⁺ interferes only with Cl^- influx, and that the negative EOG's are elicited by an increase in permeability of the olfactory receptive membrane to Na⁺ and K⁺, but not to Cl^-. From the ionic mechanism it is inferred that the negative EOG's are in most cases composites of generator and positive potentials.     

Effect of the "calcium ionophore" A-23187 on transmitter release at the frog neuromuscular junction.

The ionophore A-23187 when added to the usual Ca²⁺-Ringer at the frog neuromuscular junction has almost no effect on the frequency of miniature end-plate potentials (min.e.p.p.s). The ionophore does increase the rate of Ca²⁺ efflux from frog muscle, so it is in effective concentrations in the Ringer. When added to Ringer containing Ni²⁺ instead of Ca²⁺, the ionophore increases the min.e.p.p. frequency. We suggest that the ionophore can carry divalent cations into the terminal, but there are mechanisms to keep the Ca²⁺ low.Apparently these mechanisms are unable to rapidly eject or sequester Ni²⁺.     

Further Studies on the Roles of Sodium and Potassium in the Generation of the Electro-Olfactogram : Effects of mono-, di-, and trivalent cations

In the negative EOG-generating process a cation which can substitute for Na⁺ was sought among the monovalent ions, Li⁺, Rb⁺, Cs⁺, NH₄⁺, and TEA⁺, the divalent ions, Mg⁺⁺, Ca⁺⁺, Sr⁺⁺, Ba⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and Ni⁺⁺, and the trivalent ions, Al⁺⁺⁺ and Fe⁺⁺⁺. In Ringer solutions in which Na⁺ was replaced by one of these cations the negative EOG's decreased in amplitude and could not maintain the original amplitudes. In K⁺-Ringer solution in which Na⁺ was replaced by K⁺, the negative EOG's reversed their polarity. Recovery of these reversed potentials was examined in modified Ringer solutions in which Na⁺ was replaced by one of the above cations. Complete recovery was found only in the normal Ringer solution. Thus, it was clarified that Na⁺ plays an irreplaceable role in the generation of the negative EOG's. The sieve hypothesis which was valid for the positive EOG-generating membrane or IPSP was not found applicable in any form to the negative EOG-generating membrane. The reversal of the negative EOG's found in K⁺- , Rb⁺- , and Ba⁺⁺-Ringer solutions was attributed to the exit of the internal K⁺. It is, however, not known whether or not Cl^- permeability increases in these Na⁺-free solutions and contributes to the generation of the reversed EOG's.     

A new method for measuring the kinetics of transmembrane calcium-45 efflux in the smooth muscle of the guinea pig tanea coli.

A simple technique has been developed for resolving cellular ⁴⁵Ca efflux from intestinal smooth muscle into two exponential components. Strips of taenia coli from guinea pig are labeled with ⁴⁵Ca for three hours and then washed in an ice-cold, oxygenated medium containing 5 mM CaEGTA and 1.5 mM excess Ca²⁺ for 40 minutes in order to remove extracellularly bound label. Tissues subsequently placed in a control physiological solution at 37°C exhibit an efflux profile of ⁴⁵Ca which can be resolved into two exponential components. The apparent magnitudes and rate constants of these components are sensitive to ionic alterations of the medium and to biochemical manipulation of the Ca²⁺ compartmentalization within the cells.     

Influence of magnesium on chloride-induced calcium release in skinned muscle fibers

Chloride-induced Ca release in skinned muscle fibers was studied by measuring isometric force transients and 45Ca loss from fiber to washout solutions. Skinned fibers prepared from muscles soaked in normal Ringer solution made large force transients in 120 mM Cl solution with 5 mM ATP and 1 mM Mg, but 3 mM Mg was inhibitory. Mg inhibition was antagonized by low temperature and by Cd, agents which slow active Ca uptake by the sarcoplasmic reticulum (SR). In low Mg++, Cl stimulated rapid 45Ca release from the SR in sufficient amounts to account for the force response. The increased 45Ca release was inhibited by EGTA, suggesting that release requires free Ca under these conditions. The 45Ca initially released was partially reaccumulated later. Reaccumulation was increased in higher Mg++. These results provide additional evidence that the Ca uptake rate is an important determinant of net release, and suggest that Mg++ acts primarily on this mechanism. Skinned fibers prepared from muscles soaked in low Cl solutions could give force responses to Cl solutions with 3 mM and 6 mM Mg. This observation suggests that the Cl stimulus varies with the [Cl] gradient across the internal membranes, and supports the hypothesis that applied Cl causes membrane depolarization.     

Cyclic GMP regulation of a voltage-activated K channel in dissociated Enterocytes

Summary Enterrocytes from the intestinal epithelium of the winter flounder were isolated by collagenase digestion and incubated in flounder Ringer solution. Conventional whole-cell and amphotericin-perforated whole-cell recording techniques were used to characterize the properties of a voltage-activated K current present in dissociated cells. Resting membrane potentials and series resistances were significantly lower (from –23 to – 39 mV and 29 to 13 M, respectively) when amphotericin was used to achieve the whole-cell configuration. When cells were placed in flounder Ringer solution, held at –80 mV and subsequently stepped to a series of depolarizing voltages (from–70 to 0 mV), an outward current was observed that exhibited inactivation at voltages above –20 mV. This current was sensitive to holding potential and was not activated when the cells were held at –40 mV or above. When cells were bathed in symmetric K Ringer solution and the same voltage protocol was applied to the cell, inward currents were observed in response to the negative intracellular potentials. Reversal potentials at two different extracellular K concentrations were consistent with K as the currentcarrying ion. BaCl₂ (2 mM) and CsCl (0.5 mM) both produced voltage-dependent blockade of the current when added to the bathing solution. Charybdotoxin (300 nM extracellular concentration) completely blocked the current. The IC₅₀ for charybdotoxin was 50 nM. Cyclic. GMP inhibited the voltage-activated current in flounder Ringer and in symmetric K Ringer solution. The cyclic GMP analog, 8-Br cGMP, lowered the threshold for voltage activation and potentiated inactivation of the current at voltages above–40 mV. Previous studies with intact flounder epithelium showed that K recycling and net K secretion were inhibited by Ba²⁺ and by cGMP. We suggest that the channel responsible for the whole-cell current described in this study may be important in K recycling and secretion.     

Effects of calcium on nickel tolerance and accumulation in Alyssum species and cabbage grown in nutrient solution

Nickel (Ni) phytoextraction using hyperaccumulator plant species to accumulate Ni from mineralized and contaminated soils rich in Ni is undergoing commercial development. Serpentinite derived soils have a very low ratio of Ca/Mg among soils due the nature of the parent rock. In crop plants, soil Ca reduces Ni uptake and phytotoxicity, so it is possible that the low Ca of serpentine soils could limit hyperaccumulator plant tolerance of serpentine soils used for commercial phytomining. In this study, we investigated the effects of varied Ca concentration in the presence of high Mg characteristic of serpentine soils on Ni uptake and tolerance by serpentine-endemic species Alyssum murale Waldst. et Kit. and A. pintodasilvae T.R. Dudley in comparison with cabbage (Brassica oleracea L. var. capita) in a nutrient solution study. The levels of Ca and Mg used were based on serpentine and normal soils, and Ni was based on achieving over 1% Ni in Alyssum shoots in preliminary tests. Varied solution concentrations of Ni (31.6–1,000 μM for Alyssum, 1.0–10 μM for cabbage) and Ca (0.128–5 mM) were used in a factorial experimental design; 2 mM Mg was used to mimic serpentine soils. Alyssum spp. showed much greater tolerance to high Ni, high Mg, and low Ca solution concentrations than cabbage. For Alyssum spp., Ni induced phytotoxicity was only apparent at 1,000 μM Ni with relatively low and high Ca/Mg quotient. In the 1,000 μM Ni treatment, shoot Ni concentrations ranged from 8.18 to 22.8 g kg^?1 for A. murale and 7.60 to 16.0 g kg^?1 for A. pintodasilvae. Normal solution Ca concentrations (0.8–2 mM) gave the best yield across all Ni treatments for the Alyssum species tested. It was clear that solution Ca levels affected shoot Ni concentration, shoot yield and Ni translocation from root to shoot, but the relation was non-linear, increasing with increasing Ca up to 2 mM Ca, then declining at the highest Ca. Our results indicate that Ca addition to high Mg serpentine soils with very low Ca/Mg ratio may reduce Ni phytotoxicity and improve annual Ni phytoextraction by Alyssum hyperaccumulator species. Removal of shoot biomass in phytomining will require Ca application to maintain full yield potential.     

Ca fluxes in single twitch muscle fibers

Ca influx and efflux in single twitch muscle fibers were determined by the movement of 45Ca. The isotope was assayed by counting the center 1 cm of a fiber while it was in nonradioactive Rnger's solution. The average resting influx in 1.0 mM Ca Ringer's was 0.26 pM Ca/cm2. sec for 5 to 20 min influx periods. The average additional influx upon stimulation in 1.0 mM Ca was 0.73 pM Ca/cm2. twitch. The efflux after both resting and stimulated 45Ca influx can be described by a single exponential curve with an average time constant of 125 min. This relationship is an indication of Ca exchange with a single intracellular compartment. This compartment contains an estimated 47% of the total muscle Ca at 1.0 mM Ca. When the Ca in the Ringer was reduced to 0.5 mM Ca, both the resting and stimulated Ca fluxes decreased. When Ca was raised to 1.8 mM, the stimulated influxes increased but the resting influx did not.