Salinity-calcium interactions on growth and ionic concentration of Citrus plants

Two-year-old Navel orange scions (Citrus sinensis (L.) Osbeck) budded to either Cleopatra mandarin (C. reticulata) and Troyer citrange (C. sinensis × P. trifoliata) rootstocks were used in this experiment. Cleopatra manda in rootstock was considered more tolerant to salinity than Troyer citrange, and this property was attributed to a greater capacity to exclude chloride ions.Plants were grown under glasshouse conditions and supplied with nutrient solution containing either no or 45 mM NaCl. Calcium concentration was increased from 3 to 30 mM. Sodium, potassium, calcium and chloride concentrations in plant organs were analyzed after 90 days of treatment.Supplemental Ca was found to mitigate the adverse effects of salinity on plant growth, defoliation or leaf injury.Chemical analysis indicated that in plants grafted on Troyer citrange Ca restricted uptake and subsequent translocation of Na to the leaves and increased K concentration in both roots and leaves. However, in Cleopatra mandarin-grafted plants increasing Ca levels seemed to reduce transport of Na from roots to leaves, and Na accumulation in roots was associated with reduced concentration of K in this rootstock.Organ chloride analysis showed that Cl accumulation in leaves of plants grafted on both rootstocks was reduced when external Ca concentration increased, whereas Cl concentration in roots remained constant or increased. The data of distribution of Cl in plants showed that a high external Ca level increased Cl accumulation in the basal stem and roots, and reduced the transport of Cl from roots to leaves.     

The effect of sodicity on cotton: plant response to solutions containing high sodium concentrations

Solution culture was used to investigate whether the high solution Na concentrations and Na:Ca ratios found in sodic soils could directly affect the early growth and nutrient uptake of cotton (Gossypium hirsutum L.). Cotton was grown in nutrient solutions with three Na:Ca ratios (46:1, 4:1 and 0.2:1 mM) and three electrical conductivities (EC) (2.5, 4.25 and 6 dS m^?1) combined in a factorial design with four replicates. Most cotton growth parameters (including shoot and root dry weight, fruit number and weight) were unaffected by increasing solution EC or Na:Ca ratio, but at the highest Na concentration (56.6 mM), plant height was reduced. It was concluded that young cotton has the ability to tolerate solution Na concentrations up to those found in moderately sodic soils. Increasing solution Na:Ca increased plant root and shoot concentrations and plant accumulation for Na, and decreased them for Ca. Increasing EC also increased plant Na concentration and accumulation. Shoot K and P concentrations decreased with EC, but actually increased as the sodicity (Na:Ca ratio) of the nutrient solution increased. The results suggest that the low K and P concentrations commonly found in cotton grown in sodic soils are not a direct result of Na:Ca ratio in the soil solution.     

Sodium Extrusion and Potassium Uptake in Guinea Pig Kidney Cortex Slices

Slices from the cortex corticis of the guinea pig kidney were immersed in a chilled solution without K and then reimmersed in warmer solutions. The Na and K concentrations and the membrane potential V_m were then studied as a function of the Na and K concentrations of the reimmersion fluid. It was found that Na is extruded from the cells against a large electrochemical potential gradient. Q₁₀ for net Na outflux was ∼2.5. At bath K concentrations larger than 8 mM the behavior of K was largely passive. At the outset of reimmersion (V_m > E_K) K influx seemed secondary to Na extrusion. Na extrusion would promote K entrance, being limited and requiring the presence of K in the bathing fluid. At bath K concentrations below 8 mM, K influx was up an electrochemical potential gradient. Thus a parallel active K uptake is apparent. Q₁₀ for net K influx was ∼2.0. Dinitrophenol inhibited net Na outflux and net K influx, Q₁₀ became <1.1 for both fluxes. The ratio between these fluxes varied. Thus at the outset of reimmersion the net Na outflux to net K influx ratio was >1. After 8 minutes it was <1.     

Effect of CaSO4 and NaCl on mineral content ofLeucaena leucocephala

The effects of varying CaSO₄ and NaCl levels on the nutrient content ofLeucaena leucocephala were established by examining the concentrations of Na, Ca, Cl, K and Mg in leucaena roots, stems and leaves. Leucaena was grown in nutrient solution at four levels of CaSO₄ (0.5, 1.0, 2.5 and 5.0 mM) and NaCl (1, 25, 50 and 100 mM), in randomized blocks with five replications. Leucaena excluded sodium from stems and leaves when NaCl concentration was 50 mM or less. Sodium uptake decreased as CaSO₄ concentration increased. Calcium uptake was affected by NaCl concentration when substrate CaSO₄ concentration was 0.5 mM. At this level, 100 mM NaCl caused a marked decrease in leaf calcium and a marked increase in leaf Cl. In all other treatments, Cl uptake was not affected by CaSO₄ concentration. Potassium uptake was strongly depressed as NaCl concentration increased at low Ca concentration, but this effect was offset at high Ca. Magnesium uptake decreased as CaSO₄ levels increased.     

The Time Course of the Loss and Recovery of Contracture Ability in Frog Striated Muscle Following Exposure to Ca-Free Solutions

Using area under the contracture curve to quantitate contractures, the diffusion coefficient of calcium ions within the frog toe muscle during washout in a calcium-free solution and subsequent recovery after reintroduction of calcium to the bathing solution was calculated to be about 2 x 10^-6 cm²/sec. The diffusion coefficient measured during washout was found to be independent of temperature or initial calcium ion concentration. During recovery it was found to decrease if the temperature was lowered. This was likely due to the repolarization occurring after the depolarizing effect of the calcium-free solution. The relation between contracture area and [Ca]_o was found to be useful over a wider range than that between maximum tension and [Ca]_o. The normalized contracture areas were larger at lower calcium concentrations if the contractures were produced with cold potassium solutions or if NO₃ replaced Cl in the bathing solutions. Decreasing the potassium concentration of the contracture solution to 50 mM from 115 mM did not change the relation between [Ca]_o and the normalized area. If the K concentration of the bathing solution was increased, the areas were decreased at lower concentrations of Ca.     

Inhibition of myoblast fusion by lysosomotropic amines

Fusion of cultured chick embryo myoblasts was inhibited by treatment with several lysosomotropic amines. The concentrations required for half-maximal inhibition of fusion were approximately 2 μM for chloroquine, 30 μM for tributylamine, 3.2 mM for ammonium chloride, and 3.3 mM for methylamine. All the amines inhibited fusion appreciably at concentrations lower than those that reduced cell density. Both the rate and extent of fusion were affected by the amines, which had to be present for about 20 hr before the usual onset of fusion. Inhibition of fusion was reversible by transfer of inhibited cells to fresh medium. The amines did not cause accumulation of a nondialyzable inhibitor in the culture medium. Levels of creatine kinase increased by eight-fold or more between 18 and 65 hr in cultures treated with tributylamine or chloroquine, although this increase was not as pronounced as in control cultures. The increased creatine kinase activity in amine-treated cultures was due mainly to the BB and MB isozymes, with relatively little increase in the MM isozyme.     

Accumulation of acetylcholinesterase at newly formed nerve--muscle synapases.

The mechanism of fluid transport in the developing preimplantation mouse embryo has been studiedin vitro by inhibiting zonular tight junction formation. Compaction, the morphogenetic process permitting zonular blastomere adhesions at the 8-cell stage, was suppressed by lowering extracellular calcium (Ca). The Ca threshold required for compaction is 0.04–0.06 mM, and in concentrations above the threshold, the rate of compaction is concentration dependent, whereas the rate of blastocyst formation is not and proceeds normally. At 0.02 mM Ca, both compaction and blastocyst development are completely prevented. Although focal tight and gap junctions are present, zonular tight junctions do not develop. We conclude that Ca is required for the maximization of cell-cell contact, but not for focal tight junction and gap junction formation. When early morulae are cultured in 0.02 mM Ca, small trophoblastic vesicles develop frequently with intracellular fluid vacuoles. If early 8-cell embryos are similarly cultured, cell division continues and many blastomeres acquire small intracellular membrane-bounded vaculoes. These coalesce, the cell volume increases, and the nucleus becomes eccentrically positioned, resulting in a giant vacuolated blastomere reminiscent of a miniaturized blastocyst. We propose that (1) vacuole formation may be an exaggeration of an intermediate intracellular step in fluid transport and (2) normal cell polarity established by zonular tight junctions is required for transcellular fluid transport.     

Calcium-dependent slow potassium conductance in rat skeletal myotubes

A prolonged hyperpolarizing afterpotential (amplitude 5–20 mV, half decay time about 400 msec at 25°C) follows the action potential in myotubes and myosacs cultured from rat skeletal muscle. This slow hyperpolarizing afterpotential (hap) is mediated by an increase in membrane K conductance, because its reversal potential follows the Nernst potential for K and is not affected by other ions. The conductance increase measured during the hap (up to four times the resting input conductance) correctly predicts the time course of the slow hap. The slow hap is Ca dependent. Its amplitude decreases when bath [Ca] is lowered, and both amplitude and duration increase when bath [Ca] is raised. The slow hap is blocked by intracellular injection of the calcium chelator, EGTA. It is inhibited by solutions containing 2–4 mM manganese or 1–5 mM barium, but is not blocked by 5–20 mM tetraethylammonium. Myotubes bathed in zero [Na], high [Ca] solutions show calcium action potentials, which are inhibited by 2–10 mM manganese, nickel or cobalt. Myotubes bathed in isotonic Ca salts (or in 2 mM Ca plus 5 mM caffeine) show long-lasting (up to 10 sec) spontaneous hyperpolarizations accompanied by prolonged contractions. These hyperpolarizations are associated with a large increase in input conductance, and they reverse in sign near the K equilibrium potential. They appear to reflect activation of the Ca-sensitive K conductance by Ca released from intracellular stores. The observation that spontaneous hyperpolarizations usually occur with no prior depolarization argues that at least a portion of the slow, Ca-sensitive K conductance system can be activated by internal Ca alone, with no requirement for plasma membrane depolarization. Cultured myotubes also have a faster K conductance system, which is inhibited by 5–20 mM tetraethylammonium or 1–5 mM barium, and is not dependent on Ca for its activation.     

Effects of caffeine on crayfish muscle fibers. I. Activation of contraction and induction of Ca spike electrogenesis

Contractions are evoked in single muscle fibers of crayfish by intracellular as well as extracellular applications of caffeine. Responses to external applications in concentrations above 2 mM could be induced indefinitely. With concentrations above 5 mM the caffeine-induced responses were highly repeatable. Tensions were transient even when the caffeine remained in the bath. There was no change in resting potential, but during the contraction the effective resistance decreased about 10%. A number of factors (change in pH, Ca, K, and Cl) modified the responses. The time course of the tension was greatly prolonged when the transverse tubular system (TTS) was s swollen and was again shortened when the TTS was caused to shrink. An increased permeability to Ca induced by caffeine was evidenced by the transformation of the normally graded electrical responses to Ca spikes, which are insensitive to tetrodotoxin. The overshoot is a function of both external Ca and caffeine. A 10-fold change in Ca changed the overshoot by 19 mv in the presence of 10 mM caffeine and by 29 mv in 80 mM caffeine. The role of the increased permeability to Ca for caffeine-induced contractions will be analyzed in the accompanying paper.     

Nature of the Schwann cell electrical potential. Effects of the external ionic concentrations and a cardiac glycoside

The effects on the Schwann cell electrical potential of external ionic concentrations and of K-strophanthoside were investigated. Increasing (K)_o depolarized the cell. The potential is related to the logarithm of (K)_o in a quasi-linear fashion. The linear portion of the curve has a slope of 45 mv/ten-fold change in (K)_o. Diminutions of (Na)_o and (Cl)_o produced only small variations in the potential. Calcium and magnesium can be replaced by 44 mM calcium without altering the potential. Increase of (Ca)_o to 88 mM produced about 10 mv hyperpolarization. The cell was hyperpolarized by 11 mv and 4 mv within 1 min after applying K-strophanthoside at concentrations of 10^-3 and 10^-5 M, respectively. No variations of cellular potassium, sodium, or chloride were observed 3 min after applying the glycoside. The hyperpolarization caused by 10^-3 M K-strophanthoside was not observed when (K)_o was diminished to 1 or 0.1 mM or was increased to 30 mM. At a (K)_o of 30 mM, 10^-2 M strophanthoside was required to produce the hyperpolarizing effect. In high calcium, the cell was further hyperpolarized by the glycoside. The initial hyperpolarization caused by the glycoside was followed by a gradual depolarization and a decrease of the cellular potassium concentration. The results indicate that the Schwann cell potential of about -40 mv is due to ionic diffusion, mainly of potassium, and to a cardiac glycoside-sensitive ion transport process.     

Morphological changes of in-vitro-produced bovine blastocysts after vitrification,in-straw direct rehydration,and culture

Morphological signs of injury and regeneration following vitrification and warming of bovine embryos were studied by light and electron microscopy. In-vitro-produced Day 7 expanded blastocysts (Day 0 = day of insemination) were vitrified by a two-step equilibration method using ethylene glycol and dimethyl sulphoxide as cryoprotectants. Thawing was performed by in-straw direct rehydration, followed by in vitro culture on a granulosa cell monolayer. Embryos were processed for transmission electron microscopy immediately after warming (0 hr) as well as after 4 hr or 24 hr of culture following warming. A control group of unfrozen embryos was also processed. At 0 hr after warming, except for a rapid collapse of the blastocoele, only minor changes were detectable by stereomicroscope. However, at the ultrastructural level, signs of extensive injury were seen, including a general distension or shrinkage of mitochondria, disintegration of cell adhesions between adjacent trophoblastic cells, and complete rupture of some cells. At 4 hr, stereomicroscopic investigation revealed collapsed blastocoele and a darkened granular appearance of the cell mass. At the ultrastructural level, signs of regeneration were also observable: cells with minor injuries were re-assembled in a central area forming a small blastocoele, cell adhesion structures were re-established, and damage of mitochondria was less severe. The majority of irreversibly damaged cells or cell debris was accumulated in the perivitelline space. At 24 hr, stereomicroscopic investigation of surviving blastocysts showed no signs of the previous injury. At the ultrastructural level, cellular debris in the perivitelline space and some degenerated cells in the blastocoele were the only signs of previous injuries. In conclusion, ultrastructural investigation revealed unexpectedly extensive damage followed by a rapid regeneration and reorganization of the embryonic structure. Mol. Reprod. Dev. 48:9–17, 1997. © 1997 Wiley-Liss, Inc.     

Intracellular Concentrations of Major Ions in Rat Myelinated Axons and Glia: Calculations Based on Electron Probe X-Ray Microanalyses

Abstract: Electron probe x-ray microanalysis (EPMA) was used to measure water content (percent water) and dry weight elemental concentrations (in millimoles per kilogram) of Na, K, Cl, and Ca in axoplasm and mitochondria of rat optic and tibial nerve myelinated axons. Myelin and cytoplasm of glial cells were also analyzed. Each anatomical compartment exhibited characteristic water contents and distributions of dry weight elements, which were used to calculate respective ionized concentrations. Free axoplasmic [K⁺] ranged from ≈155 mM in large PNS and CNS axons to ≈120–130 mM in smaller fibers. Free [Na⁺] was ≈15–17 mM in larger fibers compared with 20–25 mM in smaller axons, whereas free [Cl^?] was found to be 30–55 mM in all axons. Because intracellular Ca is largely bound, ionized concentrations were not estimated. However, calculations of total (free plus bound) aqueous concentrations of this element showed that axoplasm of large CNS and PNS axons contained ≈0.7 mM Ca, whereas small fibers contained 0.1–0.2 mM. Calculated ionic equilibrium potentials were as follows (in mV): in large CNS and PNS axons, E_K = ?105, E_Na = 60, and E_Cl = ?28; in Schwann cells, E_K = ?107, E_Na = 33, and E_Cl = ?33; and in CNS glia, E_K = ?99, E_Na = 36, and E_Cl = ?44. Calculated resting membrane potentials were as follows (in mV, including the contribution of the Na⁺,K⁺-ATPase): large axons, about ?80; small axons, about ?72 to ?78; and CNS glia, ?91. E_Cl is more positive than resting membrane potential in PNS and CNS axons and glia, indicating active accumulation. Direct EPMA measurement of elemental concentrations and subsequent calculation of ionized fractions in axons and glia offer fundamental neurophysiological information that has been previously unattainable.     

Response of tomato cultivars to salinity

The responses of five tomato cultivars (L. esculentum Mill) of different degrees of salt tolerance were examined over a range of 0 to 140 mM NaCl applied for 3 and 10 weeks. Judged by both Na and Cl accumulations and maintenance of K, Ca and Mg contents with increasing salinity, the most tolerant cultivars (Pera and GC-72) showed different responses. The greater salt tolerance of cv Pera was associated with a higher Cl and Na accumulation and a lower K content in the shoot than those found in the other cultivars, typical of a halophytic response to salinity. However, the greater salt tolerance of cv GC-72 was associated with a retention of Na and Cl in the root, restriction of their translocation to the shoot and maintenance of potassium selectivity under saline conditions. The salt tolerance mechanisms that operated in the remaining cultivars were similar to that of cv GC-72, as at first they excluded Na and Cl from the shoots, accumulating them in the roots; with longer treatment, the ability to regulate Na and Cl concentrations in the plant was lost only in the most salt sensitive cultivar (Volgogradskij), resulting in a massive influx of both ions into the shoot.The salt sensitivity of some tomato cultivars to salinity could be due to both the toxic effect of Na and Cl ions and nutritional imbalance induced by salinity, as plant growth was inversely correlated with Na and Cl contents and directly correlated with K and Ca contents. This study displays that there is not a single salt tolerance mechanism, since different physiological responses among tomato cultivars have been found.     

Effect of lanthanum on ion absorption in cucumber seedling leaves

Scanning electron microscope and energy-dispersive X-ray analysis were used to study the tissular distributions of elements Na, Mg, Cl, K, Ca, Mn, and Fe in leaves of cucumber seedlings in the absence or presence of La³⁺. The results showed that the atomic percentages of Na, Mg, Cl, K, and Ca were basically reduced and those of Mn and Fe were increased in the presence of La³⁺; in addition, at 0.02 mM La³⁺, the reduced or increased degrees were higher than those at 2.0 mM La³⁺. The effects of La³⁺ on ion absorption were similar to those of Ca²⁺, suggesting that the rare earth element lanthanum affects the plant physiological mechanism by regulating the Ca²⁺ level in plant cell.     

The effects of salt depletion on blood and tissue ion concentrations in the freshwater mussel,Ligumia subrostrata (Say)

Summary The extracellular and intracellular fluid volumes of pondwater acclimatedLigumia subrostrata are equal (3.9 ml/g dry tissue). Total blood solute is 47 mOsm and is composed primarily of Na (19.1 mM), Cl (10.6 mM), HCO₃ (12.7 mM), Ca (4.3 mM), and K (0.5 mM). Major intracellular solutes are K (14.0 mM), Na (7.0 mM) and Cl (2.4 mM).L. subrostrata continuously exposed to deionized water at 20°C exhibit a maximum decrease of 23% in extracellular fluid total solute within 30 days. The maximum [Na] and [Cl] losses are 40% and 76% respectively, while [Ca] and [HCO₃] increase by 44% and 37% respectively. No apparent change in extracellular [K] occurs. Intracellular [Na] decreases 53% and [Cl] decreases 79%, but [K] declines only 15%. Intracellular fluid volume, extracellular fluid volume, and total body water decrease 17%, 31%, and 22% respectively. Inulin clearance is 0.41 ml/g dry tissue·h for pondwater acclimated mussels and declines to 0.24 ml/g dry tissue·h during salt depletion. When salt depleted mussels are returned to solutions containing Na or Cl, they experience a net uptake of salt. The accumulated ions are about equally distributed in the extra- and intracellular compartments.     

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition—Control; after 60 h—PES Day 2.5 of embryo culture; and after 96 h—PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (Control: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (Control: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification.     

Salinity and hormone interactions in affecting growth,transpiration and ionic relations ofPhaseolus vulgaris

Addition of either abscisic acid (ABA) or kinetin at 10⁻⁶ M to salinized media (20–120mM NaCl) induced remarkable effects on growth ofPhaseolus vulgaris plants. Whereas ABA inhibited the plant growth and the rate of transpiration, kinetin induced stimulation of both parameters. Moreover, ABA increased proline and phosphorus concentrations in the salinized plants whilst kinetin decreased them. ABA induced stimulation of the transport of K, Ca and Cl from root to shoot, accumulation of K, Na and Cl in root cells and inhibits the transport of Na and accumulation of Ca. Kinetin appeared to inhibit the transport and accumulation of Na and Cl, transport of K, and stimulates the accumulation of K and Ca as well as the transport of Ca. The highest influence of both ABA and kinetin was mostly observed when these hormones were used in combination with the highest concentration of NaCl (120 mM) in the medium.     

Boron and calcium increasePisum sativum seed germination and seedling development under salt stress

A beneficial effect of B and Ca application on symbiotic interaction between legume and rhizobia under saline conditions has recently been shown, suggesting conventional agricultural practices to increase crop salt tolerance. However, nothing is known about application of both nutrients on early events of legume development under salt stress, prior to the establishment of a symbiotic interaction. Therefore, the effects of different levels of B (from 9.3 to 93μM B) and Ca (from 0.68 to 5.44 mM Ca) on seed germination, root elongation, plant development, and mineral composition of pea (Pisum sativum L. cv. Argona) grown under 0 to 150 mM NaCl, were analysed. Development of plants previously germinated in the presence of salt was more impaired than that of plants put under salt stress once seeds were germinated. A NaCl concentration of 75 mM and 150 mM inhibited pea seed germination and seedling growth. The addition of either extra B or extra Ca to the germination solution prevented the reduction caused by 75 mM NaCl but not that of 150 mM NaCl. However, root elongation and plant development under salt stress (75 mM NaCl) was enhanced only by addition of both B and Ca. When plants were cultivated in the absence of external N, N content in roots and shoots originating from seeds was diminished by salt and enhanced by B and Ca, suggesting a role of these nutrients in remobilisation of seed nutrient stores. Salinity also led to an extremely high concentration of Na⁺ ions, and to a decrease of B and Ca concentrations. This can be overcome by addition of both nutrients, increasing salt tolerance of developing pea plants. The necessity of nutritional studies to increase crop production in saline soils is discussed and proposed.     

Neoaplectana carpocapsae: Nematode accumulations on chemical and bacterial gradients

That infective juveniles of the nematode Neoaplectana carpocapsae accumulated specifically around certain host insect larvae was previously reported by us. In this work, the nematode's behavior was tested on defined chemical and bacterial gradients to determine whether these stimuli could cause the phenomenon. Nematode accumulations occurred around the peaks of certain gradients and, with NaCl, the initial accumulation rate was directly proportional to concentration up to 15 mM. Since the nematode did not respond to K-acetate, K and acetate salts were used to analyze responses to different ions. Maximal accumulations were observed with 60 mM Na⁺, 6 mM Mg²⁺, 0.75–7.5 mM Ca²⁺, and 6 mM CO₃^2?. Accumulations to concentrations of Cl^?, basic pH, and gram-negative bacteria were also observed. Acidic pH, 2.5, and 7.5 mM NH₄⁺, repelled nematodes.     

Effects of caffeine on crayfish muscle fibers. II. Refractoriness and factors influencing recovery (repriming) of contractile responses

When caffeine evokes a contraction, and only then, crayfish muscle fibers become refractory to a second challenge with caffeine for up to 20 min in the standard saline (5 mM K_o). However, the fibers still respond with contraction to an increase in K_o, though with diminished tension. Addition of Mn slows recovery, but the latter is greatly accelerated during exposure of the fiber to high K_o, or after a brief challenge with high K_o. Neither the depolarization induced by the K, nor the repolarization after its removal accounts for the acceleration, which occurs only if the challenge with K had itself activated the contractile system; acceleration is blocked when contractile responses to K are blocked by reducing the Ca in the bath or by adding Mn. Recovery is accelerated by redistribution of intracellular Cl and by trains of intracellularly applied depolarizing pulses, but not by hyperpolarization. The findings indicate that two sources of Ca can be mobilized to activate the contractile system. Caffeine mobilizes principally the Ca store of the SR. Depolarizations that are induced by high K_o, by transient efflux of Cl, or by intracellularly applied currents mobilize another source of Ca which is strongly dependent upon the entry of Ca from the bathing medium. The sequestering mechanism of the SR apparently can utilize this second source of Ca to replenish its own store so as to accelerate recovery of responsiveness to a new challenge with caffeine.