Lectin and mitotic activities in root meristems of winter wheat under oryzalin effect

The effect of oryzalin, a microtubule polymerization inhibitor (10 MM), on lectin and mitotic activities (mitotic index and duration of mitotic phases) was studied in unhardened (23 degrees C) and hardened (7 days, 2-3 degrees C) winter wheat seedlings. Three wheat cultivars differing in their frost tolerance were compared. Oryzalin treatment (3 h) decreased activity of soluble lectins, increased activity of cell wall lectin mitotic index. Under these conditions, prolongation of anaphases and disappearance of telophases were detected. Plant hardening reduced the sensitivity of cell wall lectins and mitotic activity to the cytoskeleton inhibitor due, presumably, to the appearance of cold-stable microtubules. Plant growing and hardening with oryzalin stopped mitoses and caused the appearance of polyploid cells and cells with micronuclei. These abnormalities were preserved after hardening. The results obtained demonstrate an important role of microtubules in adaptation of plants to low temperature.     

Effect of cartolin on oryzalin-induced changes in lectin activity during low-temperature plant hardening

The effect of cartolin (0.33 μM), an antistress regulator of cytokinin type, on the cytoskeleton-dependent changes in lectin activity in the roots of unhardened (23°C) and cold-hardened (3°C, 7 days) 7-day-old plants of three cultivars of winter wheat (Triticum aestivum L.) was studied. In unhardened plants, cartolin increased activity of soluble and cell wall-bound lectins in a cultivar-specific mode. This is evidently important for subsequent enhancement of adaptation processes in the cell. The inhibitor of microtubule polymerization, oryzalin, reduced the activity of soluble lectins and increased that of cell wall-bound lectins. A reduced sensitivity of lectin activity to oryzalin after cartolin treatment could result from its stabilizing action on the cytoskeletal structures and on the interaction between cell-wall lectins and microtubules. The most efficient cartolin action, the suppression of oryzalin effect on lectin activity in hardened plants, was observed in the frost-sensitive wheat cultivar. It is likely that cartolin treatment is more efficient in the activation of adaptation processes occurring with the involvement of cytoskeletal structures in the cultivars of lower tolerance.     

Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin.

Maitotoxin (MTX), a putative Ca(2+) channel activator produced by the dinoflagellate Gambierdiscus toxicus showed extremely potent hemolytic and ichthyotoxic activities. Hemolysis of 1% mouse blood cell suspension in saline occurred at 15 nM of MTX. The activity was enhanced six-fold in the presence of 10 microM of Ca(2+) and completely blocked by EDTA2Na, indicating its dependency on external Ca(2+). The MTX-induced hemolysis was little affected by L-type Ca(2+) channel blockers (diltiazem, nifedipine, verapamil) but was strongly inhibited by calmodulin blockers (prenylamine and chlorpromazine) or a phospholipase A2 inhibitor (quinacrine). MTX was mimicked by a calcium ionophore, calcimycin. Based on these results, a series of cellular events triggered by MTX were presumed to occur in the following sequence: increased Ca(2+) entry in cells, activation of calmodulin, promotion of phospholipase A2 activity, and finally destruction of cell membrane resulting from hydrolysis of membrane lipids. The sensitivity of blood cells to MTX varied significantly, dependent on the animal sources. Nucleated blood cells of carps and chickens were 100 times more resistant than those of mammals. LC(50) of MTX to freshwater fish Tanichthys albonubes in Ca(2+) free media (pH 8) was 5 nM but was markedly lowered to 3 pM by raising pH to 8 and increasing Ca(2+) concentration to 2 mM. In a marine environment MTX was 2000 times more toxic to fish than 42-di-hydrobrevetoxin-B (PbTx-3), one of the best known ichthyotoxins of red-tide origins.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Cytoskeleton-induced alterations of the lectin activity in winter wheat under cold hardening and abscisic acid (ABA)

The roots and leaves of 7-day seedlings of three winter wheat cultivars differing in frost resistant were used to study changes in lectin activity under cytoskeleton modifiers (DMSO-7%; colchicine-1 m m; oryzalin-15 microm; cytochalasin B-15 microm) of non-hardened (23 degrees C) and hardened (2-3 degrees C, 3-7 day) plants. Plants were grown with ABA (30 microm) or without ABA. Pretreatment with colchicine, oryzalin [inhibitors of microtubules (MT) polymerization], cytochalasin B [inhibitor of microfilament (MF) polymerization] increased the activity of cell wall lectins, although pretreatment with DMSO (stabilizer of microtubules) decreased the activity. Both hardening and ABA decreased the effect of the cytoskeletal modifiers. These results could be explained by the appearance of tolerant MTs with less affinity. It is probable that increase in the activity of cell wall lectins may be the compensatory mechanism which stabilizes the cytoskeleton structure in conditions tending to disrupt it. The genotype with low resistance had higher sensitivity of lectin activity to cytoskeleton modifiers than the frost resistant genotype. The results suggest that leaves have more stable MTs and MFs and stronger MT-MF binding than roots.     

Inhibition of suicidal erythrocyte death by blebbistatin

Blebbistatin, a myosin II inhibitor, interferes with myosin-actin interaction and microtubule assembly. By influencing cytoskeletal dynamics blebbistatin counteracts apoptosis of several types of nucleated cells. Even though lacking nuclei and mitochondria, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include energy depletion and osmotic shock, which enhance cytosolic Ca(2+) activity with subsequent Ca(2+)-sensitive cell shrinkage and cell membrane scrambling. The present study explored the effect of blebbistatin on eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in fluorescence-activated cell sorting analysis and cytosolic Ca(2+) concentration from Fluo3 fluorescence. Exposure to blebbistatin on its own (1-50 μM) did not significantly modify cytosolic Ca(2+) concentration, forward scatter, or annexin V binding. Glucose depletion (48 h) was followed by a significant increase of Fluo3 fluorescence and annexin V binding, effects significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 10 μM). Osmotic shock (addition of 550 mM sucrose) again significantly increased Fluo3 fluorescence and annexin binding, effects again significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 25 μM). The present observations disclose a novel effect of blebbistatin, i.e., an influence on Ca(2+) entry and suicidal erythrocyte death following energy depletion and osmotic shock.     

Lanthanum inhibits steady-state turnover of the sarcoplasmic reticulum calcium ATPase by replacing magnesium as the catalytic ion

LaATP is shown to be an effective inhibitor of the calcium ATPase of sarcoplasmic reticulum because the binding of LaATP to cE.Ca2 results in the formation of lanthanum phosphoenzyme, which decays slowly. Steady-state activity of the calcium ATPase in leaky sarcoplasmic reticulum vesicles is inhibited 50% by 0.16 microM LaCl3 (15 nM free La3+, 21 nM LaATP) in the presence of 25 microM Ca2+ and 49 microM MgATP (5 mM MgSO4, 100 mM KCl, 40 mM 4-morpholinepropanesulfonic acid, pH 7.0, 25 degrees C). However, 50% inhibition of the uptake of 45Ca and phosphorylation by [gamma-32P]ATP in a single turnover experiment requires 100 microM LaCl3 (28 microM free La3+) in the presence of 25 microM Ca2+; this inhibition is reversed by calcium but inhibition of steady-state turnover is not. Therefore, binding of La3+ to the cytoplasmic calcium transport site is not responsible for the inhibition of steady-state ATPase activity. The addition of 6.7 microM LaCl3 (1.1 microM free La3+) has no effect on the rate of dephosphorylation of phosphoenzyme formed from MgATP and enzyme in leaky vesicles, while 6.7 mM CaCl2 slows the rate of phosphoenzyme hydrolysis as expected; 6.7 microM LaCl3 and 6.7 mM CaCl2 cause 95 and 98% inhibition of steady-state ATPase activity, respectively. This shows that inhibition of ATPase activity in the steady state is not caused by binding of La3+ to the intravesicular calcium transport site of the phosphoenzyme. Inhibition of ATPase activity by 2 microM LaCl3 (0.16 microM free La3+, 0.31 microM LaATP) requires greater than 5 s, which corresponds to approximately 50 turnovers, to reach a steady-state level of greater than or equal to 80% inhibition. Inhibition by La3+ is fully reversed by the addition of 0.55 mM CaCl2 and 0.50 mM EGTA; this reactivation is slow with t1/2 approximately 9 s. Two forms of phosphoenzyme are present in reactions that are partially inhibited by La3+: phosphoenzyme with Mg2+ at the catalytic site and phosphoenzyme with La3+ at the catalytic site, which undergo hydrolysis with observed rate constants of greater than 4 and 0.05 s-1, respectively. We conclude, therefore, that La3+ inhibits steady-state ATPase activity under these conditions by replacing Mg2+ as the catalytic ion for phosphoryl transfer. The slow development of inhibition corresponds to the accumulation of lanthanum phosphoenzyme. Initially, most of the enzyme catalyzes MgATP hydrolysis, but the fraction of enzyme with La3+ bound to the catalytic site gradually increases because lanthanum phosphoenzyme undergoes hydrolysis much more slowly than does magnesium phosphoenzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of TRIM on tension, intracellular calcium and nitrergic transmission in the rat anococcygeus muscle.

The effects of the putatively selective inhibitor of neuronal nitric oxide synthase (nNOS) 1-(2-trifluoromethylphenyl) imidazole (TRIM) were investigated on contractility, intracellular calcium and nitrergic relaxations in the rat anococcygeus muscle. TRIM (100-1000 microM) reduced the tension of rat anococcygeus muscles when contracted with guanethidine (10 microM) and clonidine (0.1 microM). Relaxations to TRIM persisted in the presence of the non-selective NOS inhibitor L-NAME (100 microM) and the inhibitor of soluble guanylate cyclase ODQ (1 microM). TRIM also reduced tension when muscles were contracted with phenylephrine (3 microM), noradrenaline (3 microM) or high K physiological salt solution (high KPSS; 60mM). Influx of calcium ([Ca(2+)](i)) in response to high KPSS was significantly reduced in the presence of TRIM (1mM). TRIM also inhibited the influx of (45)Ca(2+) induced by KPSS, but had no effect on the influx induced by phenylephrine (10 microM). TRIM (300 microM) had a modest, but significant, inhibitory effect on nitrergic relaxations that were evoked by electrical field stimulation (1-10 Hz, 15 V, 10s trains) in muscles contracted with guanethidine and clonidine. In contrast, L-NAME (1-100 microM) inhibited these nitrergic responses with an IC(50) of 9.31+/-0.87 microM (n=4). The results suggest that the smooth muscle relaxant effect of TRIM in the rat anococcygeus muscle may affect the entry of Ca(2+) possibly through voltage-operated calcium channels. Furthermore, the relatively modest effect of TRIM on nitrergic responses indicates that it is not a particularly reliable inhibitor of nNOS.     

Modulation of rat brain cytosolic phosphatidate phosphohydrolase: effect of cationic amphiphilic drugs and divalent cations

The effects of three cationic amphiphilic drugs on rat brain cytosolic phosphatidate phosphohydrolase and their mechanisms of action were studied utilizing membrane-bound, emulsified, and emulsified sonicated phosphatidate as substrates. With the membrane-bound substrate, chlorpromazine, desmethylimipramine, and propranolol inhibited the activity in a dose-dependent fashion with an IC50 of 30-50 microM. In the presence of the emulsified substrate, chlorpromazine was a more potent inhibitor than desmethylimipramine or propranolol but 200 microM was needed for 50% inhibition of activity. Addition of heat-inactivated microsomes to the emulsified substrate, to simulate the conditions with the membrane-bound substrate, did not alter this value. Both Mg2+ and Ca2+ stimulated the enzyme activity but only Ca2+ counteracted the effect of chlorpromazine. Kinetic studies indicate that chlorpromazine acts as a noncompetitive inhibitor of the enzyme. Emulsified sonicated phosphatidate was a good substrate at low (less than 10 microM) concentrations. It was a poor substrate at 1 mM, but at this concentration chlorpromazine stimulated the activity instead of inhibiting. This drug altered the integrity of phosphatidate vesicle membranes as visualized by electron microscopy. The different results obtained with the three types of substrate indicate the importance of the configuration of phosphatidate for the expression of enzyme activity and for its susceptibility to the action of cationic amphiphilic drugs.     

Ionomycin and 2,5'-di(tertbutyl)-1,4,-benzohydroquinone elicit Ca2+-induced Ca2+ release from intracellular pools in Physarum polycephalum

Calcium level in organelles of the slime mold Physarum polycephalum was monitored by chlortetracycline, a low-affinity calcium indicator. It was found that 2,5'-di(tertbutyl)-1,4,-benzohydroquinone (BHQ) at a concentration of 100 microM, but not the highly specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (1-10 microM), elicited calcium release from the CTC-stained intracellular calcium pool. Ionomycin also caused a calcium release (23.7+/-5.1%), which was less than that induced by BHQ (30.1+/-6.0%). Procaine (10 mM), a blocker of ryanodine receptor, completely abolished the responses to BHQ and ionomycin. Another blocker, ryanodine (100 microM), only slightly diminished the responses to ionomycin and BHQ. Apparently, BHQ and ionomycin acting as a Ca2+-ATPase inhibitor and an ionophore, respectively, elicit an increase in [Ca2+]i, which in turn triggers a calcium-induced calcium release (CICR) via the ryanodine receptor. Caffeine, an activator of ryanodine receptor, at a concentration of 25-50 mM produced a Ca2+-release (5.6-16.0%), which was not similar in magnitude to CICR. The response to 25 mM caffeine was only moderately inhibited by 25 mM procaine, and almost completely abolished by 50 mM procaine and 100 microM ryanodine.     

Chlorpromazine inhibits the calcium-mediated effects of S-100 protein(s) on assembled brain microtubule proteins, but not those on microtubule protein assembly

We have examined the S-100-chlorpromazine interplay at the level of brain microtubule proteins in vitro. The results indicate that in the presence of 0.12 M KCl and 10 microM free Ca2+ the inhibitory effect of S-100 on microtubule assembly is additive to that of chlorpromazine, but S-100 fails to potentiate the disassembling effect of 0.1 mM Ca2+ if added to assembled microtubule proteins after chlorpromazine and Ca2+, probably because of inhibition of S-100 by the phenothiazine. Chlorpromazine does not compete with S-100 for binding to purified tubulin.     

Stimulation of calcium transport of sarcoplasmic reticulum vesicles by the calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N',-tetraacetic acid

The calcium ion dependence of calcium transport by isolated sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been investigated by means of the Calcium-stat method, in which transport may be measured in the micromolar free calcium ion concentration range, in the absence of calcium buffers. At pH 7.2 and 20 degrees C, ATP, in the range 1 to 10 mM, decreased [Ca2+]0.5 from 2.0 microM to 0.3 microM and decreased Vmax of oxalate-supported transport from 0.5 to 1.3 mumol min-1 mg-1. Simultaneous measurements of transport and of ATPase activity in the range 0.8 to 10 microM free Ca2+ showed a ratio of 2.1 calcium ions translocated/molecule of ATP hydrolyzed. Transport, in the presence of 5 mM ATP, ceased when calcium ion concentration fell to 0.6 to 1.2 microM, whilst ATPase activity of 90 nmol of ATP hydrolyzed min-1 mg-1 persisted. The data obtained by the Calcium-stat method differed from those described previously using calcium buffers, in that they showed lower apparent affinities of the transport site for calcium ions, more marked sigmoidal behavior, an effect of ATP concentration on Ca2+ concentration dependence and lower ATPase activity in the absence of transport. The calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (CaEGTA) had no effect when transport was stimulated maximally at saturating free Ca2+ concentrations. However, at calcium ion levels below [Ca2+]0.5, 70 microM CaEGTA stimulated transport to rates of 20 to 45% of Vmax. Half-maximal stimulation of transport occurred at 19 microM CaEGTA. CaEGTA, 50 microM, decreased [Ca2+]0.5, determined at 5 mM ATP, from 1.3 microM to 0.45 microM. It is proposed that a ternary complex, E . Ca2+ . EGTA4-, is formed as an intermediate species during CaEGTA-stimulated calcium transport by sarcoplasmic reticulum membranes and stimulates the calcium pump at limiting free Ca2+ ion concentration.     

Stimulatory effect of regucalcin on ATP-dependent Ca(2+) uptake activity in rat liver mitochondria

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver mitochondria was investigated. The presence of regucalcin (0.1, 0.25, and 0.5 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the mitochondria. Ruthenium red (10(-5) M) or lanthanum chloride (10(-4) M), an inhibitor of mitochondrial Ca(2+) uptake, completely inhibited regucalcin (0.25 microM)-increased mitochondrial Ca(2+)-ATPase activity and (45)Ca(2+) uptake. The effect of regucalcin (0.25 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of digitonin (10(-2)%), a solubilizing reagent of membranous lipids, or vanadate (10(-5) M), an inhibitor of phosphorylation of ATPase. The activatory effect of regucalcin (0.25 microM) on Ca(2+)-ATPase activity was not further enhanced in the presence of dithiothreitol (2.5 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme, or calmodulin (0.60 microM), a modulator protein of Ca(2+) action that could increase mitochondrial Ca(2+)-ATPase activity. The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver mitochondria, and that the protein may act on an active site (SH group)-related to phosphorylation of mitochondrial Ca(2+)-ATPase.     

Brief rapid pacing depresses contractile function via Ca(2+)/PKC-dependent signaling in cat ventricular myocytes

The purpose of this study is to determine the effects of brief rapid pacing (RP; approximately 200-240 beats/min for approximately 5 min) on contractile function in ventricular myocytes. RP was followed by a sustained inhibition of peak systolic cell shortening (-44 +/- 4%) that was not due to changes in diastolic cell length, membrane voltage, or L-type Ca(2+) current (I(Ca,L)). During RP, baseline and peak intracellular Ca(2+) concentration ([Ca(2+)](i)) increased markedly. After RP, Ca(2+) transients were similar to control. The effects of RP on cell shortening were not prevented by 1 microM calpain inhibitor I, 25 microM L-N(5)-(1-iminoethyl)-orthinthine, or 100 microM N(G)-monomethyl-L-arginine. However, RP-induced inhibition of cell shortening was prevented by lowering extracellular [Ca(2+)] (0.5 mM) during RP or exposure to chelerythrine (2-4 microM), a protein kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective inhibitor of PKC-beta. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (-46 +/- 7%). Western blots indicated that cat myocytes express PKC-alpha, -delta, and -epsilon as well as PKC-beta. These findings suggest that brief RP of ventricular myocytes depresses contractility at the myofilament level via Ca(2+)/PKC-dependent signaling. These findings may provide insight into the mechanisms of contractile dysfunction that follow paroxysmal tachyarrhythmias.     

Role of regucalcin as an activator of Ca(2+)-ATPase activity in rat liver microsomes.

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. Thapsigargin (10(-6) M), a specific inhibitor of microsomal Ca(2+) pump enzyme (Ca(2+)-ATPase), clearly inhibited regucalcin (0.5 microM)-increased microsomal Ca(2+)-ATPase activity. Liver microsomal Ca(2+)-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10(-2) %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca(2+) ionophore (A23187; 10(-7) M). The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca(2+)-ATPase.     

Carbon monoxide activates KCa channels in newborn arteriole smooth muscle cells by increasing apparent Ca2+ sensitivity of alpha-subunits

Carbon monoxide (CO) is a gaseous vasodilator produced by many cell types, including endothelial and smooth muscle cells. The goal of the present study was to investigate signaling mechanisms responsible for CO activation of large-conductance Ca(2+)-activated K(+) (K(Ca)) channels in newborn porcine cerebral arteriole smooth muscle cells. In intact cells at 0 mV, CO (3 microM) or CO released from dimanganese decacarbonyl (10 microM), a novel light-activated CO donor, increased K(Ca) channel activity 4.9- or 3.5-fold, respectively. K(Ca) channel activation by CO was not blocked by 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (25 microM), a soluble guanylyl cyclase inhibitor. In inside-out patches at 0 mV, CO shifted the Ca(2+) concentration-response curve for K(Ca) channels leftward and decreased the apparent dissociation constant for Ca(2+) from 31 to 24 microM. Western blotting data suggested that the low Ca(2+) sensitivity of newborn K(Ca) channels may be due to a reduced beta-subunit-to-alpha-subunit ratio. CO activation of K(Ca) channels was Ca(2+) dependent. CO increased open probability 3.7-fold with 10 microM free Ca(2+) at the cytosolic membrane surface but only 1.1-fold with 300 nM Ca(2+). CO left shifted the current-voltage relationship of cslo-alpha currents expressed in HEK-293 cells, increasing currents 2.2-fold at +50 mV. In summary, data suggest that in newborn arteriole smooth muscle cells, CO activates low-affinity K(Ca) channels via a direct effect on the alpha-subunit that increases apparent Ca(2+) sensitivity. The optimal tuning by CO of the micromolar Ca(2+) sensitivity of K(Ca) channels will lead to preferential activation by signaling modalities, such as Ca(2+) sparks, which elevate the subsarcolemmal Ca(2+) concentration within this range.     

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.     

Parathyroid hormone activation of map kinase in rat duodenal cells is mediated by 3',5'-cyclic AMP and Ca(2+)

In a previous study, we demonstrated that parathyroid hormone (PTH) stimulates in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) isoforms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipase C and increases intracellular Ca(2+) in these cells, in the present study we evaluated the involvement of cAMP, Ca(2+) and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5-15 microM), an AC activator, or Sp-cAMP (50-100 microM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca(2+) (EGTA 0.5 mM), chelation of intracellular Ca(2+) with BAPTA (5 microM), or blockade of L-type Ca(2+)-channels with verapamil (10 microM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca(2+) mobilizing agent thapsigargin, the Ca(2+) ionophore A23187, ionomycin and membrane depolarization with high K(+). Inclusion of the calmodulin inhibitor fluphenazine (50 microM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca(2+) play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca(2+) and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.     

Isolation and characteristics of scallop sarcoplasmic reticulum with calcium transport activity.

Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year.     

Effects of Ca2+-ATPase inhibitors, ionomycin, and pharmacological modulators of ryanodine receptor on calcium release from intracellular pools and on oscillatory contractile behavior in Physarum polycephalum

Changes in calcium levels in organelles of the plasmodium of the myxomycete Physarum polycephalum were analyzed using the fluorescent calcium indicator chlortetracycline (CTC). Both the Ca2+-ATPase inhibitor 2,5;-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (100 microM) and the calcium ionophore ionomycin (1 microM) induce a significant decrease in fluorescence level (by 30%) in CTC-stained microplasmodia; this is caused by release of calcium from intracellular storage compartments. An activator of ryanodine receptors, caffeine (10-50 mM), is less effective on Ca2+ release than BHQ or ionomycin, and their inhibitor, ryanodine (100 microM), almost completely blocks the response to caffeine, but only slightly decreases the effects of BHQ or ionomycin. Procaine, another inhibitor of ryanodine receptors, at 10 mM concentration completely abolishes both the BHQ and the ionomycin responses, but 50 mM is necessary to block the effect of 25 mM caffeine. These results suggest that both the BHQ- and the ionomycin-dependent Ca2+ releases occur through the ryanodine receptor and are to be considered as calcium-induced Ca2+ release (CICR). Both the ionomycin and the BHQ responses persist in the presence of Cd2+, which blocks Ca2+ channels of the plasmalemma. In most cases, Cd2+ itself induces release of Ca2+ from the CTC-stained calcium pool; the more effective Cd2+ is, the less the following ionomycin or BHQ responses occur. This indicates that Ca2+ entry through plasmalemma plays no significant role in the ionomycin- or BHQ-evoked initiation of CICR, and that the Cd2+- and BHQ/ionomycin-depleted Ca2+ stores overlap.