Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient

The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction of the target function had been achieved in antisense transformants with multiple construct integrations. Induction conditions caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the distinctive tip-high Ca²⁺ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise regulation of apical growth, a common form of cellular proliferation. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype very similar to that of the cna-1 antisense mutants is consistent with this idea.     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca²⁺ sparks are the elementary events of intracellular Ca²⁺ release from the sarcoplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca²⁺ channel activation contributes to the genesis of spontaneous Ca²⁺ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca²⁺ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI₂ which inhibits spontaneous L-type Ca²⁺ channel activation, the rate of occurrence of spontaneous Ca²⁺ sparks was halved from 4.20 to 2.04 events/(100 μm · s), with temporal and spatial properties of individual Ca²⁺ sparks unchanged. Analysis of the Cd²⁺-sensitive spark production revealed an open probability of ~10 ^-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca²⁺ channels in resting heart cells contribute significantly to the production of spontaneous Ca²⁺ sparks.     

Isolation and characterization of the krev-1 gene, a novel member of ras superfamily in Neurospora crassa : involvement in sexual cycle progression

Genes belonging to the ras superfamily encode low-molecular-weight GTP/GDP-binding proteins that are highly conserved in wide variety of organisms. We used the polymerase chain reaction (PCR) to isolate a novel member of the ras superfamily from the filamentous fungus Neurospora crassa and obtained a mammalian Krev-1 homolog. We named the gene krev-1 and analyzed its structure and function. The krev-1 gene encodes a polypeptide of 225 amino acids, which is nearly 60% homologous to the mammalian Krev-1 p21. The krev-1 gene product (KREV1) is functionally analogous to mammalian Krev-1 p21 and Rsr1p/Bud1p, a Krev-1 homolog from the yeast Saccharomyces cerevisiae. GAL1-driven expression of KREV1 in a wild-type yeast strain resulted in a random budding pattern, as did its mammalian counterpart Krev-1 p21. We disrupted the krev-1 gene by RIP (repeat-induced point mutation), but the krev-1 disruptants showed no abnormalities. By in vitro mutagenesis, we constructed several mutant krev-1 genes (G21V, A68T, and D128A) which mimic constitutively active mutants of Ha-ras, and the krev-1 (K25N) mutant which is analogous to a dominant-negative mutant of Ha-ras. Each mutant gene was introduced into the wild-type strain and the phenotypes were analyzed. We could not observe any difference in vegetative growth between these transformants. When each strain was used as the female in mating tests, the development of perithecia from protoperithecia was inhibited in all cases. The results indicate that the krev-1 gene may be involved in sexual cycle progression. Received: 28 January 1997 / Accepted: 3 April 1997     

Involvement of Calcineurin in Ca2+ Paradox-Like Injury of Cultured Rat Astrocytes

Abstract: The Ca²⁺/calmodulin-dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused Ca²⁺ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca²⁺-mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca²⁺ challenge-induced injury in a dose-dependent manner (10⁻¹⁰–10⁻⁸ M ). Cyclosporin A at 1 µ M mimicked the effect of FK506. Rapamycin (1 µ M ) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 n M ), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca²⁺ challenge-induced increase in cytosolic Ca²⁺ level nor Na⁺-Ca²⁺ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca²⁺ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.     

Kinetic Characterization of Ca2+ Transport in Synaptic Membranes

Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Differential effects of Ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

Ａｐｏｐｔｏｓｉｓｏｒｐｒｏｇｒａｍｍｅｄｃｅｌｌｄｅａｔｈ(ＰＣＤ)ｉｓａｐｒｏｃｅｓｓｏｆｃｅｌｌｄｅｌｅｔｉｏｎｗｈｉｃｈｏｃｃｕｒｓｉｎｒｅｓｐｏｎｓｅｔｏａｎｕｍｂｅｒｏｆｃｙｔｏｔｏｘｉｃａｎｄｐｈｙｓｉｏｌｏｇｉｃａｌｌｙｒｅｌｅｖａｎｔｓｔｉｍｕｌｉ.Ｔｈｉｓｐｒｏｃｅｓｓｉｓｃｈａｒａｃｔｅｒｉｚｅｄｂｙｓｅｖｅｒａｌｅａｒｌｙｍｏｒｐｈｏｌｏｇｉｃａｌｔｅｒａｔｉｏｎｓｉｎｃｌｕｄｉｎｇｐｌａｓｍａａｎｄｎｕｃｌｅａｒｍｅｍｂｒａｎｅｂｌｅｂｂｉｎｇ.Ｅｎｄｏｇｅｎｏｕｓｅｎｄｏ…     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

铝胁迫下小麦根尖分生细胞中Ca2+分布变化

运用透射电镜细胞化学方法对铝胁迫下小麦根尖分生细胞中Ca^2 分布的变化进行了观察,在正常生长条件下,Ca^2 广泛分布于细胞质、细胞核、细胞间隙中,特别是液泡中有大量的Ca^2 沉淀颗粒;在AI^3＋胁迫条件下,细胞质、细胞核中Ca^2 沉淀颗粒明显减少,分布发生改变,细胞质中液泡增多,但其中Ca^2 沉淀颗粒明显减少。结果表明,AI^3 不但抑制了根尖细胞对Ca^2 的吸收,而且引起细胞中原有Ca^2 分布的变化,这很可能引起细胞功能的紊乱,进而影响极系的生长。     

Relation of Acetylcholine Release to Ca2+ Uptake and Intraterminal Ca2+ Concentration in Guinea-Pig Cortex Synaptosomes

[14C]Acetylcholine (ACh) release and parallel alterations in 45Ca2+ uptake and intrasynaptosomal free CA2+ concentration ([Ca2+]i) were measured in guinea-pig brain cortex synaptosomes. Depolarization by high K+ concentrations caused a rapid transient increase in Ca2+ uptake, terminating within 60 s (rate constant = 0.060 s-1; t1/2 = 11.6 s). This resulted in a rapid increase (within 1 s) in [Ca2+1]i, which then fell to a maintained but still-elevated plateau level (t1/2 for the decline was 15 s). Peaks of [Ca2+]i showed a sigmoidal dependence on depolarization, contrasting with the simple linear dependence of plateau levels of [Ca2+]i. The K+-evoked ACh release also had two phases: a fast initial increase (t1/2 = 11.3 s), which terminated within 60 s, was followed by a slow additional increase during sustained depolarizations of up to 10 min. Depolarization by veratridine led to a slow gradual increase in Ca2+ uptake (t1/2 = 130 s) over a 10-min incubation period, whereas an elevated plateau level of [Ca2+]i was achieved within 2 min (without a rapid peak elevation). The Ca2+-dependent fraction of the veratridine-evoked ACh release correlated with the increase in [Ca2+]i rather than with Ca2+ uptake. Using two different methods of depolarization partially circumvented the time limitations imposed by a buffering Ca2+ indicator and we suggest that, in the main, ACh is released in bursts associated with [Ca2+]i transients.     

Isoform-Specific Up-Regulation by Ouabain of Na+,K+-ATPase in Cultured Rat Astrocytes

Abstract: There are two α-subunit isoforms (α1 and α2) and two β-subunit isoforms (β1 and β2) of Na⁺,K⁺-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 m M ) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K⁺ and monensin (20 µ M ) mimicked the effect of ouabain on α1 mRNA. The ouabain-induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µ M ), the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetraacetoxymethyl ester (30 µ M ), and the calcineurin inhibitor FK506 (1 n M ). These findings indicate that chronic inhibition of Na⁺,K⁺-ATPase up-regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na⁺, Ca²⁺, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.     

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Role of Na+-Ca2+ Exchanger in Agonist-Induced Ca2+ Signaling in Cultured Rat Astrocytes

Abstract: We have previously demonstrated that activation of the Na⁺-Ca²⁺ exchanger in the reverse mode causes Ca²⁺ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na⁺-Ca²⁺ exchanger is involved in agonist-induced Ca²⁺ signaling in cultured rat astrocytes. The astrocytic intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased by l -glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca²⁺]_i. The Na-induced Ca²⁺ signal was also attenuated by S-nitroso-l -cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na⁺-Ca²⁺ exchanger enhanced the ionomycin-induced increase in [Ca²⁺]_i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca²⁺ signal. Furthermore, the ionomycin-induced Ca²⁺ signal was enhanced by removal of extracellular Na⁺ and pretreatment with ascorbic acid. These findings indicate that the Na⁺-Ca²⁺ exchanger is a target for NO modulation of elevated [Ca²⁺]_i and that the exchanger plays a role in Ca²⁺ efflux when [Ca²⁺]_i is raised above basal levels in astrocytes.     

Incubation of Tissue Slices in the Absence of Ca2+ and Mg2+ Can Cause Nonspecific Damage

Superfusion of striatal slices with a medium deficient in Ca2+ and Mg2+ caused a large and sustained increase in release of lactate dehydrogenase, a finding indicative of the disruption of plasma membranes. This was associated with an efflux of dopamine (DA) and the depletion of DA from the tissue. In addition, whereas DA efflux was stimulated by either D-amphetamine (10 microM) or L-glutamate (10 mM) in the absence of Ca2+, these effects were greatly reduced when Mg2+ also was withdrawn from the buffer. These results suggest that (a) incubation in a Ca2+/Mg2(+)-free buffer disrupts plasma membranes, (b) this disruption affects dopaminergic neurons as well as those of other striatal elements, and (c) the failure of a treatment to stimulate DA release in a Ca2+/Mg2(+)-free buffer cannot be used as a test of Ca2+ dependence.     

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.     

Ca2+和Sr2+诱导大肠埃希菌感受态细胞的形成及转化的影响

为研究金属离子诱导下感受态细胞形成的机理及揭示转化发生的机制,分别用Ca~(2+)和Sr~(2+)(0~140mmol/L)制备大肠埃希菌感受态细胞并转化。研究结果表明,不同浓度的Ca~(2+)和Sr~(2+)诱导的感受态细胞的效价不同,两种金属离子对大肠埃希菌细胞内外膜的通透性均有较大影响,但细胞内外膜的改变程度与转化率无直接关系;电镜结果显示,未处理的细胞呈簇聚集发生粘连现象,感受态细胞整体呈分散状态,局部发生聚集,而转化后的细胞独立存在,边缘异常清晰。     

Local Anesthetics Inhibit the Ca2+,Mg2+-ATPase Activity of Rat Brain Synaptosomes

Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R.I., Epstein P.M., Andrenyak P.M., and Feinstein M.B. (1981) Proc. Natl. Acad. Sci. USA 78, 795-799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.