The effect of divalent cations on Na+ tolerance in Charophytes. I: Char a buckellii

Abstract The comparative Na⁺ tolerance of Chora buckellii cultured in freshwater (FW) or artificial Waldsea water (AWW, which contains about 110 mol m^?3 each Na ⁺, Mg²⁺, Cl^? and SO^2-₄ was tested with respect to the external Na⁺ to Ca²⁺ ratio (Na: Ca). Fifty per cent of FW cells subjected to 70 mol m^?3 NaCl, which raised Na:Ca from 10: 1 to 700: 1 and the external osmotic pressure from 0.024 to 0.402 MPa, died within 6 d. Death was associated with the loss of Na/K selectivity, H⁺ -pump activity and turgor. Restoration of Na:Ca to 10:1 in high Na⁺ medium with CaCl₂ ensured 100% survival and maintained H⁺-pump activity and Na/K selectivity of FW cells. Turgor was regulated within 3 d with net uptake of Na ⁺, K⁺ and Cl^? in the vacuolc. Mg²⁺ was not as effective as Ca²⁺ in enhancing survival or maintaining H⁺ -pump activity and Na/K selectivity of FW cells in the presence of elevated Na⁺. However, turgor was regulated within 3 d by accumulation of Cl^? and an unknown cation in the vacuole. All AWW cells subjected to an increase of 70 mol m ^?3 NaCl, which raised Na: Ca from 16:1 to 25: 1 and the external osmotic pressure from 0.915 to 1.22 MPa, survived and maintained H ⁺ -pump activity. Turgor was regulated within 6d by accumulating Na ⁺, K⁺ and Cl^? in the vacuole. All AWW cells subjected to 70molm^?3 NaCl in a medium in which Na:Ca was equal to 700:1 survived and maintained H ⁺ -pump activity, but showed loss of Na/K selectivity. Turgor was regulated with an unknown osmoticum(a) within 6 d.     

Calcium-salinity interactions affect ion transport in Chara corallina

Detached internodes of Chara corallina survived in solutions containing 100 mol m^?3 NaCl when the external concentration of Ca²⁺ was greater than 1 mol m^?3. Na⁺ influx was roughly proportional to external Na⁺ up to 100 mol m^?3 NaCl. Na⁺ influx involved two components: a Ca²⁺-insensitive influx which allowed the passage of Na⁺ independently of external Ca²⁺; and a Ca²⁺-inhibitable mechanism where Na⁺ influx was inversely proportional to external Ca²⁺. The Ca²⁺-inhibitable Na⁺ influx was similar to the Ca²⁺-inhibitable K⁺ influx. Mg²⁺ and Ba²⁺ were able to substitute for Ca²⁺ in partially inhibiting Na⁺ influx in the absence of external Ca²⁺. The effect of Ca²⁺ appears specific to Na⁺ and K⁺ influx since the effects of a Ca²⁺-free solution on the influx of some other cations, anions and neutral compounds is small. It is suggested that Na⁺ influx via the Ca²⁺-inhibitable mechanism represents Na⁺ leakage through K⁺ channels and that cell death at high salinity occurs due to a cytotoxic Na⁺ influx via this mechanism.     

Effects of salinity and turgor on calcium influx in Chara

Measurements were made of the influx of ⁴⁵Ca into internodal cells of Chara corallina in solutions containing high concentrations of NaCl. Increasing salinity in the range 4–100mol m^?3 NaCl resulted in a doubling of Ca²⁺ influx at the plasmalemma. A time-course of Ca²⁺ influx in 50 mol m^?3 NaCl, 0.5mol m^?3 CaCl₂ showed that while influx at the plasmalemma increased only 1.5-fold, influx to the vacuole increased by up to 15-fold. This was interpreted as being due to inhibition of active Ca²⁺ efflux from the cell. The stimulation of Ca²⁺ influx by increasing salinity appeared to be principally a response to reduced turgor since similar stimulations were obtained when turgor was reduced by NaCl, Na₂SO₄ or mannitol. When cells were plasmolysed Ca²⁺ influx increased by 10–20-fold. The increased permeability was relatively specific for Ca²⁺ and was inhibitable by La³⁺. Survival of cells in high salt conditions was increased by 30 mmol m^?3 La³⁺, which inhibited Ca²⁺ influx. Paradoxically, survival can also be extended by increasing external Ca²⁺ which leads to a higher influx. Therefore, it seems unlikely that the ameliorative effect of Ca²⁺ on the sensitivity of plants to high NaCl is mediated by Ca²⁺ entry across the plasmalemma. It seems more likely that the principal role of Ca²⁺ under these conditions is exerted externally through the control of membrane voltage and permeability.     

Differential effects of Na+, Mg2+, K+, Ca2+ and osmotic stress on the wild type and the NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii

In order to identify physiological components that contribute to salinity tolerance, we compared the effects of Na⁺, Mg²⁺ and K⁺ salts (NaCl, Na₂SO₄, MgCl₂, MgSO₄, KCl and K₂SO₄), Ca₂₊ (CaSO₄), mannitol and melibiose on the wild type and the single-gene NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii. Compared with gametophytic growth of the wild type, stl2 showed a low level of tolerance that was restricted to Na⁺ salts and osmotic stress. stl2 exhibited high tolerance to both Na⁺ and Mg²⁺ salts, as well as to osmotic stress. In response to short-term exposure (3 d) to NaCl, accumulation of K⁺ and Na⁺ was similar in the wild type and stl1. In contrast, stl2 accumulated higher levels of K⁺ and lower levels of Na⁺. Ca²⁺ supplementation (1.0 mol m^?3) ameliorated growth inhibition by Na⁺ and Mg²⁺ stress in wild type and stll, but not in stl2. In addition, under Na⁺ stress (175 mol m^?3) wild-type, stll and stl2 gametopbytes maintained higher tissue levels of K⁺ and lower levels of Na⁺ when supplemented with Ca²⁺ (1.0 mol m^?3). stl2 gametophytes were extremely sensitive to K⁺ supplementation. Growth of stl2 was greater than or equal to that of the wild type at trace concentrations of K⁺ but decreased substantially with increasing K⁺ concentration. Supplementation with K⁺ from 0 to 1.85 mol m^?3 alleviated some of the inhibition by 75 mol m^?3 NaCl in the wild type and in stl1. In stl2, growth at 75 mol m^?3 NaCl was similar at 0 and 1.85 mol m^?3 K⁺ supplementation. Although K⁺ supplementation above 1.85 mol m^?3 did not alleviate inhibition of growth by Na⁺ in any genotype, stl2 maintained greater relative tolerance to NaCl at all K⁺ concentrations tested.     

Control of sodium influx by calcium and turgor in two charophytes differing in salinity tolerance

The effects of Ca²⁺ and cell turgor on Na⁺ influx were examined in two charophytes, lamprothamnium papulo-SUM (salt-tolerant) and Chara corallina (salt-sensitive), to try to identify causes of salinity toxicity. Mortality was associated with Na+ influx, with the two species showing similar sensitivities to high Na⁺ influx. In Lamprothamnium, toxic influxes of Na⁺ occurred at much higher external Na⁺ concentrations than in Chara. The differences in Na⁺ influx at the same Na⁺ concentration were not due to different responses to external Ca²⁺. Lamprothamnium adjusts its turgor in response to increasing NaCl whereas Chara cannot. In solutions of KC1 up to at least 200 mol m_-3, however, Chara regulated turgor, and when KC1 was subsequently replaced with NaCl, Na+ influx was low and similar to that in Lamprothamnium at the same Na* concentration. Chara cells which were not turgor-adjusted in KCI had Na⁺ influxes 2-5-fold higher than the turgid cells. Thus, it appears that turgor is a major determinant of Na⁺ influx, and therefore of cell survival. We found no evidence that the mechanism of Na⁺ influx in Chara is different from that in Lamprothamnium. Higher susceptibility of Chara to NaCl seems to result from inability to regulate turgor, in turn leading to toxic Na⁺ influx.     

Pacemaking mechanism of the afterdischarge of the ovulation hormone-producing caudo-dorsal cells in the gastropod mollusc Lymnaea stagnalis

The ovulation hormone-producing caudo-dorsal cells (CDC) of Lymnaea stagnalis have three states of excitability (active, inhibited, and resting), which are related to the egg-laying cycle. Active state CDC produce a firing pattern of prolonged spiking activity (1 spike/2 s), which in the animal occurs shortly before egg laying. In preparations it is evoked as an afterdischarge upon repetitive stimulation of CDC. The afterdischarge is not synaptically driven, but rests on a pacemaking mechanism. CDC are silent in the inhibited and resting states, which follow egg laying. In these states the membrane potential is mainly dependent on [K⁺]₀. In the active state the ratio of the K⁺, Na⁺, and Ca²⁺ permeabilities has changed considerably, probably resulting from an increased permeability to Na⁺ and Ca²⁺. The firing rate in the afterdischarge is dependent on the membrane potential, which is confirmed experimentally by varying [K⁺]₀.[Na⁺]₀ and [Ca²⁺]₀ directly influence the firing rate. Firing stops in Na⁺-free saline, but is enhanced by Ca²⁺-free or high-Mg²⁺ saline. TTX does not affect firing. Relatively high concentrations of Co²⁺ and La³⁺ (2 × 10^?3M) strongly inhibit CDC. Regular firing can be changed into bursting by various means, such as high K⁺ or addition of 1 mM Ba²⁺. Bursting normally occurs at the beginning of the afterdischarge. Postburst hyperpolarizations are reduced in Ca²⁺-free saline and by low Co²⁺ (10^?4-5 10^?4M). Active CDC are driven by a pacemaking mechanism constituted by a voltage-dependent Na⁺/Ca²⁺ channel and a Ca²⁺-dependent K⁺ channel, thus resembling that of bursting pacemakers. The pacemaking mechanism is inactive in the resting and inhibited state.     

Sodium transport measured in plasma membrane vesicles isolated from wheat genotypes with differing K+/Na+ discrimination traits

Right-side-out plasma membrane vesicles were isolated from wheat roots using an aqueous polymer two-phase system. The purity and orientation of the vesicles were confirmed by marker enzyme analysis. Membrane potential (Ψ)-dependent ²²Na⁺ influx and sodium/proton (Na⁺/ H⁺) antiport-mediated efflux across the plasma membrane were studied using these vesicles. Membrane potentials were imposed on the vesicles using either K⁺ gradients in the presence of valinomycin or H⁺ gradients. The ΔΨ was quantified by the uptake of the lipophilic cation tetraphenylphosphonium. Uptake of Na⁺ into the vesicles was stimulated by a negative ΔΨ and had a K_m for extrav-esicular Na⁺ of 34.8 ± 5.9 mol m³. The ΔΨ-dependent uptake of Na⁺ was similar in vesicles from roots of hexaploid (cv. Troy) and tetraploid (cv. Langdon) wheat differing in a K⁺/Na⁺ discrimination trait, and was also unaffected by growth in 50 mol m^?3 NaCl. Inhibition of ΔΨ-dependent Na⁺ uptake by Ca²⁺ was greater in the hexaploid than in the tetraploid. Sodium/proton antiport was measured as Na⁺-dependent, amiloride-inhibited pH gradient formation in the vesicles. Acidification of the vesicle interior was measured by the uptake of ¹⁴C-methylamine. The Na⁺/H⁺ antiport had a K_m, for intravesicular Na⁺ of between 13 and 19 mol m^?3. In the hexaploid, Na⁺/H⁺ antiport activity was greater when roots were grown in the presence of 50 mol m^?3NaCl, and was also greater than the activity in salt-grown tetraploid wheat roots. Antiport activity was not increased in a Langdon 4D chromosome substitution line which carries a trait for K⁺/Na⁺ discrimination. It is concluded that neither of the transport processes measured is responsible for the Na⁺/K⁺ discrimination trait located on the 4D chromosome of wheat.     

Turgor regulation in a brackish water charophyte,Lamprothamnium succinctum

Internodal cells of a brackish water charophyte,Lamprothamnium succinctum (A. Br. in Ash.) R.D.W. regulate the turgor pressure in response to changes in both the cellular and the external osmotic pressures. During turgor regulation upon hypotonic treatment, net effluxes of K⁺ and Cl⁻ from the vacuole, membrane depolarization, a transient increase in the electrical membrane conductance and a transient increase in concentration of cytoplasmic Ca²⁺ are induced. Activation of the plasmalemma Ca²⁺ channels and the Ca²⁺-controlled passive effluxes of K⁺ and Cl⁻ through the plasmalemma ion channels are postulated.     

高盐生境中乌拉尔甘草盐离子分泌与积存格局的研究

采用植物水培方法,以乌拉尔甘草为研究材料,用不同浓度(0、80、160、320mmol·L~(-1))NaCl溶液胁迫处理乌拉尔甘草幼苗3周后,分析其叶片表面盐离子(K~+、Ca~(2+)、Na+)分泌速率的差异,并采集盐化低地草甸重盐土生境中2年生乌拉尔甘草植株,应用ICP-AES测定其不同部位(根、根状茎、茎、老叶和幼叶)中的盐离子(K~+、Na~+、Ga~(2+)、Mg~(2+))含量,探究盐离子在乌拉尔甘草叶片上的分泌格局以及盐离子在植株体内的积存格局,为完善甘草耐盐机理的研究提供依据。结果显示:(1)随着盐胁迫浓度的升高,乌拉尔甘草叶片上K~+、Ca~(2+)、Na+的分泌速率均呈增加趋势,且Na~+的分泌速率远远大于Ca~(2+)和K+的分泌速率。(2)在乌拉尔甘草各部位中,K+的积存量从大到小依次为:幼叶根根状茎茎老叶;Na~+在各个部位的积存量都十分有限,且无论地下部分还是地上部分,差异均不大;Ca~(2+)积存量由大到小依次为:老叶幼叶茎根状茎根,且老叶中Ca~(2+)的积存量显著高于其它部位。研究认为,在重盐碱地生境中,K+主要积存在幼叶中,Ga~(2+)主要积存在老叶中,植株体内各个部位Na~+的积存量很低,乌拉尔甘草表现出明显的拒Na现象;叶片分泌的主要盐离子为Na~+;乌拉尔甘草通过泌盐的方式将Na+排出体外,从而有效降低Na~+在体内的积存,这是其能够在重盐碱地生存生长的重要原因。     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Ionic and osmotic components of salt stress specifically modulate net ion fluxes from bean leaf mesophyll

Ionic mechanisms of salt stress perception were investigated by non‐invasive measurements of net H⁺, K⁺, Ca²⁺, Na⁺, and Cl^? fluxes from leaf mesophyll of broad bean (Vicia faba L.) plants using vibrating ion‐selective microelectrodes (the MIFE technique). Treatment with 90 m M NaCl led to a significant increase in the net K⁺ efflux and enhanced activity of the plasma membrane H⁺‐pump. Both these events were effectively prevented by high (10 m M ) Ca²⁺ concentrations in the bath. At the same time, no significant difference in the net Na⁺ flux has been found between low‐ and high‐calcium treatments. It is likely that plasma membrane K⁺ and H⁺ transporters, but not the VIC channels, play the key role in the amelioration of negative salt effects by Ca²⁺ in the bean mesophyll. Experiments with isotonic mannitol application showed that cell ionic responses to hyperosmotic treatment are highly stress‐specific. The most striking difference in response was shown by K⁺ fluxes, which varied from an increased net K⁺ efflux (NaCl treatment) to a net K⁺ influx (mannitol treatment). It is concluded that different ionic mechanisms are involved in the perception of the ‘ionic’ and ‘osmotic’ components of salt stress.     

High salinity tolerance in the stl2 mutation of Ceratopteris richardii is associated with enhanced K+ influx and loss

The roles of K⁺ uptake and loss in the salinity response of the wild type and the salt-tolerant mutant stl2 of Ceratopteris richardii were studied by measuring Rb⁺ influx and loss and the effects of Na⁺, Mg²⁺, Ca²⁺ and K⁺-transport inhibitors. In addition, electrophysiological responses were measured for both K⁺ and Rb⁺ and for the effects of Na⁺ and NH₄⁺ on subsequent K⁺-induced depolarizations. stl2 had a 26–40% higher uptake rate for Rb⁺ than the wild type at 0.5–10 mol m^?3 RbCl. Similarly, membrane depolarizations induced by both RbCl and KCl were consistently greater in stl2. In the presence of 0–180 mol m^?3 NaCl, stl2 maintained a consistently greater Rb⁺ influx than the wild type. stl2 retained a greater capacity for subsequent KCl-induced depolarization following exposure to NaCl. Five mol m^?3 Mg²⁺ decreased Rb⁺ uptake in stl2; however, additional Mg²⁺ up to 40 mol m^?3 did not affect Rb⁺ uptake further. Ca²⁺ supplementation resulted in a very minor decrease of Rb⁺ uptake that was similar in the two genotypes. Tetraethylammonium chloride and CsCl gave similar inhibition of Rb⁺ uptake in both genotypes, but NH₄Cl gave substantially greater inhibition in the wild type than in stl2. NH₄Cl resulted in a greater membrane depolarization in the wild type and the capacity for subsequent depolarization by KCl was markedly reduced. stl2 exhibited a higher Independent loss of Rb⁺ than the wild type, but, in the absence of external K⁺, loss of Rb⁺ was equivalent in the two genotypes. Since constitutive K⁺ contents are nearly identical, we conclude that high K⁺ influx and loss exact a metabolic cost that is reflected in the inhibition of gametophytic growth. Growth inhibition can be alleviated by reduced supplemental K⁺ or by treatments that slightly reduce K⁺ influx, such as moderate concentrations of Na⁺ or Mg²⁺. We propose that high throughput of K⁺ allows maintenance of cytosolic K⁺ under salt stress and that a high uptake rate for K⁺ results in a reduced capacity for the entrance and accumulation of alternative cations such as Na⁺ in the cytosol.     

Mechanisms underlying turgor regulation in the estuarine alga Vaucheria erythrospora (Xanthophyceae) exposed to hyperosmotic shock

Aquatic organisms are often exposed to dramatic changes in salinity in the environment. Despite decades of research, many questions related to molecular and physiological mechanisms mediating sensing and adaptation to salinity stress remain unanswered. Here, responses of Vaucheria erythrospora, a turgor‐regulating xanthophycean alga from an estuarine habitat, have been investigated. The role of ion uptake in turgor regulation was studied using a single cell pressure probe, microelectrode ion flux estimation (MIFE) technique and membrane potential (E_m) measurements. Turgor recovery was inhibited by Gd³⁺, tetraethylammonium chloride (TEA), verapamil and orthovanadate. A NaCl‐induced shock rapidly depolarized the plasma membrane while an isotonic sorbitol treatment hyperpolarized it. Turgor recovery was critically dependent on the presence of Na⁺ but not K⁺ and Cl^? in the incubation media. Na⁺ uptake was strongly decreased by amiloride and changes in net Na⁺ and H⁺ fluxes were oppositely directed. This suggests active uptake of Na⁺ in V. erythrospora mediated by an antiport Na⁺/H⁺ system, functioning in the direction opposite to that of the SOS1 exchanger in higher plants. The alga also retains K⁺ efficiently when exposed to high NaCl concentrations. Overall, this study provides insights into mechanisms enabling V. erythrospora to regulate turgor via ion movements during hyperosmotic stress.     

A New Turgor/Membrane Potential Probe Simultaneously Measures Turgor and Electrical Membrane Potential

Abstract: A new combined turgor/membrane potential probe (T-EP probe) monitored cell turgor and membrane potential simultaneously in single giant cells. The new probe consisted of a silicone oil-filled micropipette (oil-microelectrode), which conducted electric current. Measurements of turgor and hydraulic conductivity were performed as with the conventional cell pressure probe besides the membrane potential. In internodal cells of Chara corallina, steady state turgor (0.5-0.7 MPa) and resting potentials (-200 to ?220 mV) in APW, and hydraulic conductivity (0.07 to 0.21 × 10～⁵ m s^?1 MPa^?1) were measured with the new probe, and cells exhibited healthy cytoplasmic streaming for at least 24 h during measurements. When internodal cells of Chara corallina were treated with 30, 20, 10, and 5 mM KCI, turgor responded immediately to all concentrations, and the osmotic changes in the medium were measured. Action potentials, which brought the membrane potential to a steady depolarization that measured the concentration difference of K⁺ in the medium, were induced in a concentration — dependent delay and occurred only 30, 20, and 10 mM of KCl. When the solution was changed back to APW, the repolarization of membrane potential consisted of a quick and a following slow phase. During the quick phase, which took place immediately and lasted 1 to 3 min, the plasma membrane remained activated. The membrane was gradually deactivated in the slow phase, and entirely deactivated when the membrane potential recovered to the resting potential in APW. Although the activated plasma membrane was permeable to K⁺, no major ion channels were activated on the tonoplast, and therefore, internodal cells of Chara corallina did not regulate turgor when osmotic potential changed in the surrounding medium.     

Binding of L-[3H]Glutamate to Fresh or Frozen Synaptic Membrane and Postsynaptic Density Fractions Isolated from Cerebral Cortex and Cerebellum of Fresh or Frozen Canine Brain

Abstract: Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L-[³H]glutamate binding sites. It was found that there was a concentration of L-glutamate binding sites in CTX-PSD and CL-PSD over the respective membrane fractions, and the B_max value of CL-PSD (92.0 pmol/mg protein) was about three times that of CTX-PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL-PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L-glutamate binding to canine CTX-SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl^? increased the number of L-glutamate binding sites and the effect was enhanced by Ca²⁺; Ca²⁺ alone had no significant effect; (b) the Cl^?/Ca²⁺ -sensitive binding sites were abolished by 2-amino-4-phosphonobutyrate (APB) or freezing and thawing: (c) the effect of Na⁺ ion was biphasic: low concentration of Na⁺ (< 5 mM) decreased Cl^?7Ca²⁺ -de-pendent L-glutamate binding sites, whereas at higher concentrations of Na⁺ the binding of glutamate was found to increase either in the presence or absence of Ca²⁺ and Cl^?. In addition, the K⁺ ion (50 mM) was found to decrease the Na⁺-independent and Cl^?/Ca^2--independent binding of L-glutamate to fresh CTX-SPM by 18%, but it decreased the Na^?-dependent and Cl^?/Ca²⁺-independent L-glutamate binding by 93%; in the presence of Cl, ^?/Ca²⁺, the K⁺ ion decreased the Na⁺-dependent binding by 78%. Freezing and thawing of CTX-SPM resulted in a 50% loss of the Na⁺-dependent L-glutamate binding sites assayed in the absence of Ca²⁺ and Cl^?. The CL-SPM fraction showed similar ion dependency of L-glutamate binding except for the absence of Na^?-dependent glutamate binding sites. The CTX-PSD fraction contained neither Na⁺-dependent nor APB (or Cl^?/Ca²⁺)-sensitive L-glutamate binding sites and its L-glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L-Glutamate binding to CTX-SPM or CTX-PSD fraction was not affected by pretreatment with 10 mM L-glutamate, nor by simultaneous incubations with calmodulin. Also, phosphorylation of CTX-SPM or CTX-PSD fraction, whether incubated simultaneously or after removal of the phosphorylating reagents, had no effect on binding of L-glutamate. Furthermore, binding of L-glutamate to CTX-SPM or CTX-PSD was found to have no significant effect on subsequent phosphorylation of the fractions. Treatment of the CTX-PSD fraction with 0.5% deoxycholate, 1.0% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl, and 1.0 M KCl removed the L-glutamate receptors from the PSD by 25%, 44%, 40%, 8%, and 11%. respectively. The respective percentages of total protein solubilized by these reagents were similar, indicating no preferential dissociation of the receptors, and suggesting that the L-glutamate receptor is an intrinsic PSD component. The present findings, together with the earlier ones showing the presence of γ-aminobutyric acid and flunitrazepam binding sites, of the Ca²⁺-dependent K⁺ channel, and of the voltage-dependent Ca²⁺ channel proteins in the isolated PSD fraction, suggest that many, if not all, neurotransmitter receptor proteins and ion channel proteins are anchored in the PSD at the synapse, and thus the PSD may play an important role in neurotransmission at the postsynaptic site.     

Effects of interrupted K+ supply on growth and uptake of K+, Ca2+, Mg2+ and Na+ in spring wheat

Effects of interrupted K⁺ supply on different parameters of growth and mineral cation nutrition were evaluated for spring wheat (Triticum aestivum L. cv. Svenno). K⁺ (2.0 mM) was supplied to the plants during different periods in an otherwise complete nutrient solution. Shoot growth was reduced before root growth after interruption in K⁺ supply. Root structure was greatly affected by the length of the period in K⁺ -free nutrient solution. Root length was minimal, and root branching was maximal within a narrow range of K⁺ status of the roots. This range corresponded to cultivation for the last 1 to 3 days, of 11 in total, in K⁺ -free nutrient solution, or to continuous cultivation in solution containing 0.5 to 2 mM K⁺. In comparison, both higher and lower internal/external K⁺ concentrations had inhibitory effects on root branching. However, the differing root morphology probably had no significant influence on the magnitude of Ca²⁺, Mg²⁺ and Na⁺ uptake. Uptake of Ca²⁺ and especially Mg²⁺ significantly increased after K⁺ interruption, while Na⁺ uptake was constant in the roots and slowly increased in the shoots. The two divalent cations could replace K⁺ in the cells and maintain electroneutrality down to a certain minimal range of K⁺ concentrations. This range was significantly higher in the shoot [110 to 140 μmol (g fresh weight)^?1] than in the root [20 to 30 μmol (g fresh weight)^?1]. It is suggested that the critical K⁺ values are a measure of the minimal amount of K⁺ that must be present for physiological activity in the cells. At the critical levels, K⁺ (⁸⁶Rb) influx and Ca²⁺ and Mg²⁺ concentrations were maximal. Below the critical K⁺ values, growth was reduced, and Ca²⁺ and Mg²⁺ could no longer substitute for K⁺ for electrostatic balance. In a short-term experiment, the ability of Ca²⁺ to compete with K⁺ in maintaining electroneutrality in the cells was studied in wheat seedlings with different K⁺ status. The results indicate that K⁺, which was taken up actively and fastest at the external K⁺ concentration used (2.0 mM), partly determines the size of Ca²⁺ influx.     

Effect of Divalent Cations on Na+ Permeability of Chara corallina and Freshwater Grown Chara buckellii

The effect of elevated Na⁺ concentration on Na⁺ permeability(P_Na) and Na⁺ influx in the presence of two levels of externaldivalent cations was determined in Chara corallina and freshwater-culturedChara buckellii. When Na⁺ in the medium was increased from 1.0to 70 mol m^–3, Na⁺ influx increased in both species ifCa²⁺ was low (0.1 mol m–3). If Ca²⁺ was increased to 7.0mol m^–3 when Na⁺ was increased, Na⁺ influx remained atthe low control level in C. corallina, and showed only a temporaryincrease in C. buckellii. Mg²⁺ was a better substitute for Ca²⁺in C. buckellii than in C. corallina. Na⁺ permeability data suggest that when the external Ca²⁺ concentrationis low, P_Na does not increase in the presence of elevated NaCl;the increase in Na⁺ influx appears to be due to the increasein external Na⁺ concentration alone. Ca^{2 +} supplementation appearsto decrease P_Na whereas supplemental Mg²⁺ has no effect. Na⁺ effluxes were computed from previously determined net fluxesand the influxes. It was found that for both species, fluxesin both directions were stimulated in response to all experimentaltreatments, but Na⁺ influx always exceeded efflux. This resultedin net Na⁺ accumulation in the vacuoles of both species. The results are discussed with reference to net flux and electrophysiologicaldata obtained previously under identical conditions, as wellas the comparative salinity tolerance of both species and theNa⁺/divalent cation ratio. Key words: Na⁺ influx, Na⁺ tolerance, membrane potential, permeability, Chara     

The modulation of the calcium pump of human red cells by Na+ and K+

1. The sidedness of Ca²⁺-pump activation by Na⁺ and K⁺ was studied by atomic absorption spectrophotometry in human erythrocyte ghosts, which had been prepared in dextran solutions and resealed to alkali cations. 2. When ghosts were incubated in an all-choline medium, the increase in Na_i⁺ elicited an inhibitory-stimulatory effect on Ca²⁺ extrusion. By contrast, only a stimulatory action was induced when choline was replaced by Na₀⁺. 3. A dual effect on active Ca²⁺ efflux was also produced by increasing K_i⁺ or K₀⁺. The biphasic response to the latter, however, was absent from high-K⁺ ghosts. Furthermore, the stimulation obtained at high K₀⁺ was additive to that elicited by K_i⁺. 4. The results suggest that Na⁺ and K⁺ stimulate the Ca²⁺ pump of human red cells through two different mechanisms. The first one appears to be an electric coupling between Ca²⁺ efflux and the external activating cation. The other seems associated with the molecular reactions of the Ca²⁺-pump protein.     

Calmodulin levels in radish (Raphanus sativus L.) seeds germinating at low calcium availability induced by EGTA treatments

Incubation of radish (Raphanus sativus L.) seeds in the presence of 1 or Smol m^?3 Ca-EGTA, which increased Ca²⁺ activity in the incubation medium (c. 0.24 or 0.37 mol m^?3 at 24 h with respect to c. 0.13 mol m^?3 in the control), did not affect germination, the restoration of K⁺ net influx, the increase in DNA and RNA levels or protein synthesis. Incubation in 1 mol m^?3 Na-EGTA, which reduced Ca²⁺ activity in the incubation medium (20 mmol m^?3 at 24 h), decreased the total Ca²⁺ level in embryo axes (-21%), but only slightly inhibited the increase in fresh weight without affecting the restoration of K⁺ net influx, the increase in DNA and RNA levels or protein synthesis. In the presence of 5 mol m^?3 Na-EGTA (Ca²⁺ activity in the incubation medium was 0.6 mmol m^?3), the decrease in the total Ca²⁺ level was greater (c. -27%) and the increases in fresh weight, DNA and RNA were inhibited by about 50, 39 and 40%, respectively. These results indicate that increased Ca²⁺ availability does not affect germination and suggest that the effect of Na-EGTA, at least up to 5 mol m^?3, is a result of an induction of Ca²⁺ deficiency. The amount and specific activity of calmodulin (CaM) present in the soluble fraction (100 000g) of radish embryo axes greatly increased during the first 24 h of incubation (c. 5-fold and 7-fold, respectively). This increase was very similar in the Ca-EGTA-treated seeds but was inhibited (c. -38%) by 1 mol m^?3 Na-EGTA, even if the increases in DNA and RNA levels and protein synthesis were not significantly reduced. The lower amount of CaM after 24 h of incubation in 1 mol m^?3 Na-EGTA (c. -30%) was due to a reduction in the fraction of CaM bound to a proteinaceous CaM inhibitor present in radish seeds [M. Cocucci & N. Negrini (1988) Plant Physiology 88, 910–914] and not involved in the metabolic reactivation of the seed. These results suggest that the level of CaM is controlled by Ca²⁺ availability and that the CaM inhibitor has a role in controlling the amount of Ca-CaM available for the Ca-CaM-dependent enzymes.