Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

Neutral organic solute effects on the activity of the plasma membrane Ca2+-ATPase

We have compared effects of dimethylsulfoxide (Me₂SO) and two polyols on the Ca²⁺-ATPase purified from human erythrocytes. As studied under steady-state conditions over a broad solute concentration range and temperature, Me₂SO, glycerol, and xylitol do not inhibit the Ca²⁺-ATPase activity; this is in contrast to numerous other organic solutes that we have investigated. Under specific experimental conditions, Me₂SO (but not glycerol) substantially increases Ca²⁺-ATPase activity, suggesting a possible facilitation of enzyme oligomerization. The activation is more pronounced at low Ca²⁺ concentrations. In contrast to glycerol, Me₂SO shows no protective effect on enzyme structure as assessed by determining residual Ca²⁺-ATPase activity after exposing the enzyme to thermal denaturation at 45°C. Under these conditions several other organic solutes strongly enhance the denaturating effect of temperature. Because of the temperature dependence of its effect on the Ca²⁺-ATPase activity we believe that Me₂SO activates the Ca²⁺-ATPase by indirect water-mediated interactions.     

Ca2+ transport and chemoreception in Paramecium

Intracellular Ca²⁺ levels in Paramecium must be tightly controlled, yet little is understood about the mechanisms of control. We describe here indirect evidence that a phosphoenzyme intermediate is the calmodulin-regulated plasma membrane Ca²⁺ pump and that a Ca²⁺-ATPase activity in pellicles (the complex of cell body surface membranes) is the enzyme correlate of the plasma membrane pump protein. A change in Ca²⁺ pump activity has been implicated in the chemoresponse of paramecia to some attractant stimuli. Indirect support for this is demonstrated using mutants with different modifications of calmodulin to correlate defects in chemoresponse with altered Ca²⁺ homeostasis and pump activity.Abbreviations EGTA ethyleneglycol tetra-acetate - ER endoplasmic reticulum - IBMX isobutyl methylxanthine - I _che index of chemokinesis - Mops 3-[N-morpholino] propanesulfonic acid - PEI phosphoenzyme intermediate - STEN sucrose, TRIS, EDTA, sodium chloride - TCA trichloroacetic acid - TRIS tris[hydroxymethyl] aminomethane     

Ca2+-independent form of protein kinase C may regulate Na+ transport across frog skin

Summary Activators of protein kinase C (PKC) stimulate Na^– transport (J _Na) across frog skin. We have examined the effect of Ca²⁺ on PKC stimulation ofJ _Na. Both the phorbol ester 12-O-tetradecanoylglycerol (DiC₈) were used as PKC activators. Blocking Ca²⁺ entry into the cytosol (either from external or internal stores) reduced the subsequent natriferic effect of the PKC activators. This negative interaction did not simply reflect saturation of activation of the apical Na⁺ channels, since the stimulations produced by blocking Ca²⁺ entry and adding cyclic AMP were simply additive.The Ca²⁺ dependence of the natriferic effect could have reflected either a direct action of cytosolic Ca²⁺ on PKC or an indirect action on the final receptor site (the Na⁺ channel). To distinguish between these possibilities, the TPA- and phospholipid-dependent kinase activity of broken-cell preparations was assayed. The kinase activity was not stimulated by physiological levels of Ca²⁺, and in fact was inhibited at millimolar concentrations of Ca²⁺.We conclude that the effects of Ca²⁺ on the natriferic response to PKC activators are indirect. Reducing cytosolic uptake of Ca²⁺ may have stimulated Na⁺ transport by a chemical modification of the apical channels observed in other tight epithelia. The usual stimulation of Na⁺ transport produced by PKC activators in frog skin may reflect the operation of a nonconventional form of PKC. This enzyme is Ca²⁺ independent and seems related to thenPKC or PKC observed in other systems.     

Prostaglandin synthetase activity of goat vesicular microsomes: Co-factor requirement and effect of calcium ions

The effects of several co-factors and bivalent cations on the activity of prostaglandin synthetase isolated from goat seminal vesicles were studied. Ca²⁺ appears to play a regulatory role in the biosynthesis of prostaglandin E₂ by goat vesicular microsomes as the normal parabolic time course of synthesis changed to a sigmoid curve in the presence of 4 mM Ca²⁺ and to nearly a hyperbolic pattern when the microsomes were preincubated with the metal ions. The Ca²⁺ modulated reaction showed increased rate of prostaglandin E₂ synthesis only when the period of incubation was extended beyond 30 min. The co-factor requirement of the goat enzyme was similar to that of the bovine and ovine prostaglandin synthetase systems.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+)

The plasma membrane (Ca²⁺+Mg²⁺)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca²⁺ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca²⁺ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca²⁺-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca²⁺ is related to opposite long-term hydration effects on the substrate binding domain and the Ca²⁺ binding domain.     

Effect of Cu2+-ascorbic acid on lipid peroxidation,Mg2+-ATPase activity and spectrin of RBC membrane and reversal by erythropoietin

The effect of erythropoietin (Ep), a glycoprotein hormone, has been studied on lipid peroxidation induced by Cu²⁺ and ascorbate in vitro, Mg²⁺ ATPase activity and spectrin of RBC membrane. Our present investigation reveals that Cu²⁺ and ascorbic acid increases lipid peroxidation of RBC membrane significantly. It has further been observed that under the same experimental condition spectrin, a major cytoskeleton membrane protein, and Mg²⁺-ATPase activity of RBC membrane decrease significantly. However, exogenous administration of Ep completely restores lipid peroxidation and Mg²⁺-ATPase activity and partially recovers spectrin of RBC membrane.     

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca-ATPase

Membrane Ca²⁺-ATPase activity was stimulated in vitro separately by T₄ (10⁻¹⁰ M) and by epinephrine (10⁻⁶ M). In the presence of a fixed concentration of T₄, additions of 10⁻⁸ and 10⁻⁶ M epinephrine reduced the T₄ effect on the enzyme. β-Adrenergic blockade with propranolol (10⁻⁶ M) prevented stimulation by epinephrine of Ca²⁺-ATPase activity, but did not prevent the suppressive action of epinephrine on T₄-stimulable Ca²⁺-ATPase. In contrast α₁-adrenergic blockade with unlabelled prazosin restored the effect of T₄ on Ca²⁺-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulations by T₄ of red cell Ca²⁺-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (K_d, 1.2 × 10⁻⁸ M; B_max, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains α₁-radrenergic receptor sites that are capable of regulating Ca²⁺-ATPase activity.     

Receptor-mediated supra-additive activation of guinea pig superior cervical ganglion adenylate cyclase: Role of Mn2+ ions and calmodulin

The effects of Mn²⁺ and calmodulin were studied on the basal and agonist-modulated adenylate cyclase activity of the guinea pig superior cervical ganglion. The divalent cation strongly stimulates the basal and agonist-modulated enzyme in a concentration-dependent manner. Moreover, in the presence of Mn²⁺ the inhibitory effects of high GTP concentrations and of D-Ala²-Met-enkephalinamide on adenylate cyclase are eliminated, while the stimulation exerted by prostaglandin E₂ and the supra-additive activation of the enzyme by the combination of the two drugs are unaffected. In EGTA-washed, calmodulin-depleted membrane preparations, Mn²⁺ still activates the cyclase but the enkephalin inhibition and the superactivation of the enzyme induced by the combination of opiate and prostaglandin are lost, both in the absence and in the presence of the cation. Reconstituting the depleted membranes with exogenous Ca²⁺/calmodulin fully restored the enzyme responsivity to the combination and, partially, to the enkephalin. The findings suggest the existence in the guinea pig superior cervical ganglion of both the calmodulin-sensitive and differently regulated calmodulin-insensitive adenylate cyclase.     

Platelet Ca2+ handling in essential hypertension: Role of a plasma ouabain-like factor

A humoral ouabain-like plasma factor has been observed in patients with essential hypertension (EHT). In the present study, we hypothesized that this humoral factor might be responsible for the elevated cytosolic free calcium concentrations [Ca²⁺]_i seen in these patients. Patients with mild to moderate EHT and their normotensive first degree blood relatives (NTBR) participated in the study. Platelet Na⁺, K⁺-ATPase activity was assayed in EHT patients and their NT first-degree relatives. To confirm the ouabain-like activity in plasma from EHT patients, control platelets were incubated with EHT and NTBR plasma and their Na⁺, K⁺-ATPase activity was measured. In addition, the effect of EHT plasma on platelet⁴⁵Ca-uptake was studied. Thein vitro effects of ouabain (10 ΜM) on (i)⁴⁵Ca-uptake and (ii) [Ca²⁺]_i response in control platelets were also observed. A decreased Na⁺K⁺-ATPase activity (P< 0.05) was observed in platelet membranes from EHT patients. Incubation of control platelets with EHT plasma decreased their Na⁺, K⁺-ATPase activity (P< 0.01) and increased their⁴⁵Ca-uptake (P< 0.05). C-18 Sep-Pak filtered hypertensive plasma extracts (containing the ouabain-like fraction) also decreased Na⁺, K⁺-ATPase activity (P< 001) in control platelet membranes.In vitro incubation of control platelets with ouabain increased⁴⁵Ca-uptake (P< 005) and [Ca²⁺]_i response (P< 0.05) in these platelets. Thus it appears that an ouabain-like factor in the EHT plasma may contribute to the elevated platelet [Ca²⁺]_i observed in EHT patients.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

The alteration of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl₄) solution was investigated. Rats received a single oral administration of CCl₄ (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl₄ administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity, indicating that the increased Ca²⁺ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity increased by CCl₄ administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca²⁺-Mg²⁺)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl₄-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver obtained from CCl₄-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca²⁺ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl₄ administration.     

Cr3+和Cd2+对普通小球藻生长及抗氧化酶活性的影响

[目的] 为探究重金属对淡水绿藻生长的影响。[方法] 选取对水质检测具有明显指示作用的普通小球藻（Chlorella vulgaris）为实验材料,CdCl₂·2H₂O和CrCl₃·7H₂O提供重金属离子,探究不同浓度Cr³⁺和Cd²⁺在单一和复合胁迫下对藻细胞浓度、叶绿素a及相关抗氧化酶活性的影响。[结果] 随着Cr³⁺和Cd²⁺浓度不断增加,藻细胞浓度呈先增长后下降趋势;叶绿素a含量呈现先下降后升高再下降的现象,浓度为1 mg/L的单一和复合胁迫下有最大值,且毒性作用表现为Cr³⁺ < Cd²⁺ < Cr³⁺+Cd²⁺;与藻细胞膜相关的丙二醛（MDA）和过氧化氢（H₂O₂）含量随着重金属离子浓度的增大而增长;重金属离子浓度低于10 mg/L时对藻细胞内抗氧化酶系统中的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）表现为促进作用,而大于10 mg/L时具有抑制作用。[结论] 结果表明在单一或复合重金属胁迫下,普通小球藻会充分调动与抗逆性相关的酶来维持自身的正常生长。     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Ethanol modulates calmodulin-dependent Ca2+-activated ATPase in synaptic plasma membranes

The effects of ethanol in vitro on calmodulin-dependent Ca²⁺-activated ATPase (CaM–Ca²⁺-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca²⁺-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca²⁺-ATPase in synaptic plasma membranes is believed to be the Ca²⁺ pump controlling free Ca²⁺ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase adenosine triphosphatase - CaM calmodulin - CaM–Ca²⁺-ATPase calmodulin-dependent Ca²⁺-activated ATPase - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - EtOH ethanol - Hepes N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SPM synaptic plasma membranes - TFP trifluoperazine - Tris tris(hydroxymethyl)aminomethane - K_m Michaelis constant - T_d transition temperature - V_max maximum velocity     

Failure of short term stimulation to reduce sarcoplasmic reticulum Ca2+-ATPase function in homogenates of rat gastrocnemius

To examine the effect of short term intense activity on sarcoplasmic reticulum (SR) Ca²⁺ sequestering function, the gastrocnemius (G) muscles of 11 anaesthetized male rats (weight, 411±8 g,X±SE) were activated using supramaximal, intermittent stimulation (one train of 0.2 msec impulses per sec of 100 msec at 100 Hz). Homogenates were obtained from stimulated white (WG-S) and red (RG-S) tissues, assayed for Ca²⁺ uptake and maximal Ca²⁺ ATPase activity and compared to contralateral controls (WG-C, RG-C). Calcium uptake (nmoles/mg protein/min) determined using Indo-l and at [Ca²⁺]_f concentrations between 300–400 nM was unaffected (p>0.05) by activity in both WG (6.14+0.43 vs 5.37+0.43) and RG (3.21+0.18 vs 3.07+0.20). Similarly, no effect (p>0.05) of contractile activity was found for maximal Ca²⁺ ATPase activity (mole/mg protein/min) determined spectrophotometrically in RG (0.276+0.03 vs 0.278+0.02). In WG, Ca²⁺ ATPase activity was 15% higher in WG-S compared to WG-C (0.412+0.03 vs 0.385+0.04). Repetitive stimulation resulted in a reduction in tetanic tension of 74% (p<0.05) by 2 min in the G muscle. By the end of the stimulation period, ATP concentration was reduced (p<0.05) by 57% in the WG and by 47% in the RG. These results indicate that the repeated generation of maximal tetanic force, at least for short term periods, need not adversely affectin vitro homogenate determination of Ca²⁺ sequestering function in spite of severe alterations in energy potential and that some other mechanism must be involved to explain the depression in Ca²⁺ uptake and Ca²⁺ ATPase activity previously noted with short term intense exercise.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots

The plasmalemma vesicles isolated from cucumber and maize roots were used to study the effect of Cu²⁺ and Cd²⁺ on the hydrolytic and proton pumping activities of ATPase. In vivo application of metal ions to the plant growth solutions resulted in stimulation of the proton transport in maize. In cucumber roots the action of metals was not the same: cadmium stimulated the H⁺ transport through plasmalemma whereas Cu²⁺ almost completely inhibited it. Copper ions decreased the hydrolytic activity of H⁺-ATPase in cucumber, without any effect on this activity in membranes isolated from maize roots. The effect of cadmium on the hydrolytic activities was opposite: ATP-hydrolysis activity in plasmalemma was not altered in cucumber, whereas in maize its stimulation was observed. The amount of accumulated metals was not the main reason of different influence of metals on H⁺-ATPase activity in tested plants. In in vitro experiments Cu²⁺ inhibited H⁺ transport in the cucumber, to a higher degree than Cd²⁺ and both metals did not change this H⁺-ATPase activity of plasmalemma isolated from corn roots. Cu²⁺ added into the incubation medium reduced the hydrolytic activity of ATPase in the plasma membrane isolated from cucumber as well as from corn roots. Cd²⁺ diminished the hydrolytic activity of ATPase in cucumber, and no effect of Cd²⁺ in the plasmalemma isolated from corn roots was found. Our results indicated different in vitro and in vivo action of both metals on H⁺-ATPase and different response of this enzyme to Cu²⁺ and Cd²⁺ in maize and cucumber.