Volume decrease of glycerinated polymorphonuclear leukocytes induced by ATP and Ca : 1. Resemblances to actomyosin contraction

The addition of 1 mM ATP to rabbit peritoneal polymorphonuclear leukocytes suspended in a solution of glycerol causes a decrease in their volume by 4–17% within 3 min. The suspending medium must not only contain glycerol but be of low ionic strength for ATP to be effective. Divalent cations are also required. Ca²⁺ present alone can sustain the volume increase induced by ATP; in the presence of low concentrations of Ca²⁺, Mg²⁺ is also effective but not to the same extent as Ca²⁺. When the cell volume is contracted by the ATP in the presence of Ca²⁺ the addition of EGTA induces a reexpansion of the volume.The organic mercurial, salyrgan prevents the ATP induced reduction in the volume but ouabain has no effect. Guanosine triphosphate (GTP), uridine triphosphate (UTP), and adenosine diphosphate (ADP) can also decrease the volume of the glycerinated leukocytes but to a distinctly lesser extent than ATP. Adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) are without significant effect.The results indicate that the volume decrease caused by exogenous ATP is unlikely to be a passive osmotic or an active, ouabain-sensitive process. The similarities to the interaction of ATP with actomyosin suggest that the volume decrease might be a result of the contraction of the leukocyte actomyosin by ATP.     

ATP und Ca2+ induzierte actomyosinartige Kontraktion glycerinisierter Makronuklei

Summary The addition of 2 mM-3 mM ATP to macronuclei ofParamecium bursaria suspended in a glycerol buffer medium causes a decrease in their volume up to 23% within 3 minutes. The infiltration medium must not only contain Ca²⁺, but must also be of low ionic strength for ATP to be effective. A slow, careful exchange of the glycerol medium for the contraction solution is also necessary. Ca²⁺ present alone in the standard contraction buffer can likewise induce a limited volume decrease; in the presence of low concentrations of Ca²⁺, Mg²⁺ shows no detectable effect on glycerinated nuclei. When the nuclear volume has been reduced by ATP in the presence of Ca²⁺, the addition of EGTA induces a reexpansion of the nuclei. Salyrgan, an organic mercurial, either prevents or abolishes the ATP-induced contraction. Other nucleotide triphosphates such as guanosine triphosphate (GTP), inosine triphosphate (ITP) or uridine triphosphate (UTP) likewise induce a volume decrease of the glycerinated macronuclei, but to a distinctly lesser extent than ATP.The results indicate that the volume decrease caused by the ATP-contraction solution is not a passive osmotic process. The resemblance to actomyosin contractions suggests that the volume decrease reported here might also be the result of the reaction of nuclear actomyosin and ATP.

     

Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), as measured with the Ca²⁺-dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P₁ receptors in mediating this response. The increase in [Ca²⁺]_i was reduced in calcium-free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca²⁺]_i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5′-[γ-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) ～ adenosine 5′-[β-thio]diphosphate ? adenosine 5′-[αβ-methylene] triphosphate, adenosine 5′-[βγ-imido]triphosphate. This profile is not characteristic of either the P_2X or the conventional P_2Y receptors. The Ca²⁺ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose-dependent manner, suggesting receptor coupling to phosphatidylinositol-specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non-P_2Y nucleotide receptors (P_2N) that mediate calcium influx and the accumulation of inositol phosphates. © 1993 Wiley-Liss, Inc.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.     

Effect of bivalent cations on the adenosine triphosphatase of actomyosin and its modification by tropomyosin and troponin

1. After removal of tropomyosin and troponin from the `natural'' actomyosin complex, the adenosine triphosphatase activity of the resulting `desensitized'' actomyosin is stimulated to the same extent by various bivalent cations with an ionic radius in the range 0·65–0·99å when tested at optimum concentration of the metal ion in the presence of 2·5mm-ATP at low ionic strength and pH7·6. Under identical conditions the adenosine triphosphatase activity of myosin alone is stimulated to an appreciable extent only by Ca²⁺ (ionic radius 0·99å). 2. Tropomyosin narrows the range of size of the stimulatory cations by inhibiting specifically the adenosine triphosphatase activity of `desensitized'' actomyosin when stimulated by Ca<sup>2+</sup> or the slightly smaller Cd<sup>2+</sup> (ionic radius 0·97å). Tropomyosin has no effect on the adenosine triphosphatase activity of `desensitized'' actomyosin when stimulated by the smaller cations, nor on the Ca²⁺-activated adenosine triphosphatase activity of myosin alone. 3. The adenosine triphosphatase activity of the `natural'' actomyosin system (containing tropomyosin and troponin) stimulated by the smallest cation, Mg²⁺ (ionic radius 0·65å), is low when the system is deprived of Ca²⁺ but high in the presence of small amounts of Ca²⁺. This sensitivity to Ca²⁺ seems to be a unique feature of the Mg²⁺-stimulated system. 4. The changes in specificity of the myosin adenosine triphosphatase activity in its requirement for bivalent cations caused by interaction with actin, tropomyosin and troponin primarily concern the size of the metal ions. The effects on enzymic properties of myofibrils due to tropomyosin and troponin can be demonstrated at low and at physiological ionic strength.     

Characterization of ATP release from cultures enriched in cholinergic amacrine‐like neurons

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine‐like neurons. Depolarization of the cells with KCl, or activation of α‐amino‐3‐hydroxy‐ 5‐methyl‐4‐isoxazole‐propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca²⁺ dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and ω‐Agatoxin IVA, but not by ω‐Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca²⁺ influx necessary to trigger the release of ATP occurs in part through the L‐ and the P/Q types of voltage‐sensitive Ca²⁺ channels (VSCC), but not through N‐type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist, showing that the release is tonically inhibited by the adenosine A₁ receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 340–348, 1999     

The relaxing protein system of striated muscle. Resolution of the troponin complex into inhibitory and calcium ion-sensitizing factors and their relationship to tropomyosin

1. A method involving isoelectric precipitation and chromatography on SE-Sephadex (sulphoethyl-Sephadex) is described for the preparation of the troponin complex free of tropomyosin from low-ionic-strength extracts of natural actomyosin and myofibrils. 2. Purified troponin complex required tropomyosin to inhibit the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin in the presence of ethanedioxybis(ethylamine)tetra-acetate. An upper limit of 35000 for the `molecular weight' of the troponin complex was derived from the amounts required to bring about 50% of the maximum inhibition of the Mg²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin of known concentration. 3. In the presence of dissociating reagents the troponin complex could be dissociated into inhibitory and Ca²⁺-sensitizing factors, which could be isolated separately on SE-Sephadex. The inhibitory factor inhibited the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin independently of the concentration of free Ca²⁺ in the medium. 4. The Ca²⁺-sensitizing factor changed its electrophoretic mobility on polyacrylamide gel in the presence of ethanedioxybis(ethylamine)tetra-acetate. It formed a complex with the inhibitory factor at low ionic strength and the original biological activity of the troponin complex could be restored on mixing the inhibitory factor with the Ca²⁺-sensitizing factor in the ratio of about 3:2. 5. Evidence is presented indicating that the ability of tropomyosin preparations to restore relaxing-protein-system activity to the troponin complex and their inhibitory effect on the Ca²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin are two properties of different stability to preparative procedures and tryptic digestion. This suggests that the relaxing protein system of muscle may contain another as yet uncharacterized component.     

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) in C₆ glioma cells with an EC₅₀ of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca²⁺]_i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca²⁺ and Na⁺ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca²⁺ channels (Co²⁺, Mn²⁺, La³⁺, or Cd²⁺). In Ca²⁺-free medium, ATP caused only a transient increase in [Ca²⁺]_i as opposed to a sustained [Ca²⁺]_i increase in normal medium. The ATP-induced elevation of [Ca²⁺]_i was resistant to Na⁺ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La³⁺. The differences in the characteristics of ATP-caused P₁ hydrolysis and [Ca²⁺]_i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca²⁺ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P₁-purinergic receptors, the C₆ glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na⁺]_o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - IP₄ inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C     

Dynamic affinity chromatography of heavy meromyosin subfragment-1

Heavy meromyosin subfragment-1 and its trinitrophenylated derivative 3ave been chromatographed on immobilized ATP, ADP and adenosine 5′-(β,γ-imino)triphosphate affinity chromatography columns, in the presence and in the absence of Mg²⁺ or Ca²⁺. Splitting of bound ATP was followed by using [γ-^{3 2}P]ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5′(β,γ-imino)triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the P_i-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.     

Ca2+ responses to acetylcholine and adenosine triphosphate in the otocyst of chick embryo

The action of acetylcholine and adenosine triphosphate (ATP) on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in the otocyst epithelium of embryonic day 3 chicks with Ca²⁺-sensitive fluorescence measurements. Increases in [Ca²⁺]_i were evoked by the bath application of acetylcholine (1 μM or higher). The rise in [Ca²⁺]_i was due to the release of Ca²⁺ from intracellular Ca²⁺ stores, since the Ca²⁺ response occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolisehd the response to 10 μM acetylcholine; muscarine and carbamylcholine (100 μM each) evoked Ca²⁺ rises. Increases in [Ca²⁺]_i were also evoked by the bath application of ATP (10 μM or higher). The Ca²⁺ rise by ATP was evoked even in a Ca²⁺-free medium. Adenosine (500 μM) did not cause any Ca²⁺ response. Suramin and reactive blue 2 (200 μM each) completely blocked the Ca²⁺ response to 500μM ATP. Uridine triphosphate (500 μM) caused comparable Ca²⁺ responses with those to 500 μM ATP. These results suggested the involvement of P_2U purinoceptors. The potentiation of Ca²⁺ rise was observed when acetylcholine and ATP were co-applied at submaximal concentrations (10 μM and 100 μM, respectively). We conclude that undifferentiated cells in the otocyst epithelium have CaCa²⁺ mobilizing systems activated by acetylcholine and ATP. © 1995 John Wiley & Sons, Inc.     

Lysosomal exocytosis of ATP is coupled to P2Y2 receptor in marginal cells in the stria vascular in neonatal rats

Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague–Dawley rats were established. P2Y₂ receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immunolabelled in marginal cells of the stria vascular. We found that 30 μM ATP and 30 μM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Ca²⁺, whereas the response was suppressed by 100 μM suramin, 10 μM 1-(6-(17β-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 μM 2-aminoethoxydiphenyl borate (2-APB) and 5 μM thapsigargin (TG), thus indicating that ATP coupled with the P2Y₂R-PLC-IP3 pathway to evoke Ca²⁺ release from the endoplasmic reticulum (ER). Incubation with 200 μM Gly-Phe-β-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca²⁺]_I; this effect was partly inhibited by P2Y₂R-PLC-IP3 pathway antagonists. After pre-treatment with 5 μM TG, [Ca²⁺]_i was significantly lower than that after treatment with P2Y₂R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca²⁺ triggers Ca²⁺ release from ER Ca²⁺ stores. Baseline [Ca²⁺]_i declined after treatment with the Ca²⁺ chelator 50 μM bis-(aminophenolxy) ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 μM ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 μM vacuolin-1. Release of ATP and β-hexosaminidase both increased after treatment with 200 μM GPN and 5 μM TG, but decreased after incubation with 50 μM BAPTA-AM, 4 IU/ml apyrase and 5 μM vacuolin-1. We suggest that ATP triggers Ca²⁺ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca²⁺ triggers Ca²⁺ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca²⁺ signals indirectly via the P2Y₂R-PLC-IP3 pathway.     

P2X<Subscript>7</Subscript> Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X₇ receptor activated a reversible, Ca²⁺-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C_m) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5′-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C_m without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2′,3′-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2′4′-disulfonic acid, adenosine 5′-triphosphate-2′3′-dialdehyde, and Cu²⁺. The capacitance decrease persisted in Na⁺-, Ca²⁺- and Cl⁻-free external saline or with Ca²⁺-free pipette solution. It is concluded that ATP activation of the inotropic P2X₇ receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca²⁺ or influx of Na⁺ or Ca²⁺ through the receptor-gated channel.     

Effect of extracellular adenosine 5′-triphosphate on Ca2+ efflux from freshly isolated adult rat cardiomyocytes

The effect of extracellular adenosine 5′-triphosphate (ATP) on Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes was examined. ATP stimulated the efflux of ⁴⁵Ca²⁺ from the cells in a concentration-dependent manner (0.01–1 mM). The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by adenosine-5′-O-(3-thiotriphosphate) (ATP-γs) and α,β-methylene-ATP and adenosine 5′-diphosphate, but not by adenosine 5′-monophosphate and adenosine. The ATP-stimulated ⁴⁵Ca²⁺ efflux was not affected by deprivation of the extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. These results indicate that ATP stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane P₂ purinoceptors which may couple to Na⁺/Ca²⁺ exchange.     

Effects of xenon on intracellular Ca2% release in human endothelial cells

Using human endothelial cells loaded with the Ca^2% indicator Fura2 the effects of xenon on changes in intracellular Ca^2% were studied. The basal level of intracellular Ca^2% is not affected upon incubation of the cells in buffer saturated either with 100% xenon or with 70% xenon/30% air, a concentration which corresponds in humans to the minimum alveolar concentration necessary to induce anesthesia in 50% of patients. A defined cellular response such as the Ca^2% change induced by application of adenosine triphosphate (ATP) makes it possible to study the signalling chain between the stimulus and the various forms of Ca^2% response. ATP induces a typical Ca^2% fingerprint composed of an internal Ca^2% release consisting of several oscillations plus an additional Ca^2%-induced Ca^2% influx from the outside. The latter is absent in Ca^2%-free medium. When cells are incubated with xenon, only the first part of the ATP-induced Ca^2% response is found corresponding to the internal release of Ca^2%; the subsequent Ca^2%-induced Ca^2% influx does not take place. If xenon is removed, a fast recovery is observed and the cells again show both parts of the Ca²⁺ response. Such selective inhibition of Ca²⁺-induced Ca²⁺ influx is not obtained when xenon is replaced by N₂; the ATP response of the cell remains the same as that of untreated cells. Similar effects of xenon treatment can also be observed when the cells are treated with thapsigargin, a specific inhibitor of the SERCA systems. The Ca²⁺-induced Ca²⁺ release is almost completely suppressed in the presence of xenon. We conclude that xenon may act on the cellular level on defined sites of the mechanisms regulating the Ca²⁺-release-activated Ca²⁺ channels of the plasma membrane and that this property may be related to its anesthetic effect.     

Role of mitochondrial calcium uniporter‐mediated Ca2+ and iron accumulation in traumatic brain injury

Previous studies have suggested that the cellular Ca²⁺ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca²⁺ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca²⁺ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca²⁺ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca²⁺ and iron concentrations, but suppressed the Ca²⁺ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca²⁺ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.     

Properties of the calcium-sensitive components of bovine arterial actomyosin.

A calcium-sensitive actomyosin was prepared from bovine aortic muscularis. Calcium binding to this arterial actomyosin was measured using a centrifugation method. The amount of bound calcium necessary for full activation of the arterial actomyosin adenosine triphosphatase was approximately 36 μmol of Ca²⁺/kg of aorta. Calcium stimulation of the actomyosin ATPase could be prevented by lowering the free magnesium from 7 to 1 mm. However, calcium binding to the actomyosin increased slightly with a reduction of free magnesium levels. Positive cooperativity was evident in the sequence of reactions beginning with the binding of calcium and ending with the hydrolysis of ATP. However, there was no evidence for the cooperativity occurring at the initial calcium-binding step.     

Effects of mobile phone emissions on human red blood cells

Raman spectroscopy was performed on GSM 900 and 1800 MHz mobile phone signal exposed red blood cells (RBCs). The observed changes in the Raman spectra of mobile signal exposed RBCs compared to unexposed control suggest reduced hemoglobin-oxygen affinity for the exposed cells. The possible mechanism may involve activation of the voltage gated membrane Ca²⁺ channels by the mobile phone emissions resulting in an increase in the levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) in cells via altered metabolic activities. Further studies carried out with fluorescent Ca²⁺ indicator confirmed increased intracellular Ca²⁺ level in the exposed cells. Since intracellular ATP level influences the shape and mechanics of RBCs, exposed cells were studied using diffraction phase microscopy and optical tweezers. Detectable changes in shape and mechanical properties were observed due to mobile signal exposure.     

The reaction between actomyosin and adenosine triphosphate

1. A study is made of the effect of adenosine triphosphate (ATP) upon the viscosity of solutions of actomyosin in 0.5 M KCl. 2. The observed effects are discussed in terms of an initial drop of the viscosity (viscosity response) and its subsequent slow reversal (recovery effect). The latter is ascribed to a decrease in the ATP concentration through enzymatic hydrolysis. 3. The recovery effect is inhibited by Mg, activated by Ca, in accordance with the effect of these ions on the activity of myosin-ATPase. 4. The viscosity response is not inhibited, probably promoted by Mg. It is not promoted, probably inhibited by Ca. 5. The viscosity response is induced not only by ATP, but to a certain extent also by inosinetriphosphate, inorganic triphosphate, and inorganic pyrophosphate, not by adenosine diphosphate or monophosphate. 6. The viscosity response could be obtained with enzymatically inactive myosin. 7. It is concluded that the effect of ATP upon myosin does not depend on its enzymatic hydrolysis.     

The effect of tropomyosin on the adenosine triphosphatase activity of desensitized actomyosin

1. Tropomyosin preparations of the Bailey type, and those prepared in the presence of dithiothreitol to prevent oxidation of protein thiol groups, inhibit the Ca²⁺-activated adenosine triphosphatase (ATPase) of desensitized actomyosin by up to 60%. 2. The inhibitory activity of myofibrillar extracts and tropomyosin survives various agents known to denature proteins but to the action of which tropomyosin is unusually stable, namely heating at 100° and mild tryptic digestion. It is destroyed by prolonged treatment with trypsin. 3. The ethylenedioxybis-(ethyleneamino)tetra-acetic acid (EGTA)-sensitizing factor present in extracts of natural actomyosin and myofibrils could be selectively destroyed, leaving unchanged the inhibitory effect on the Ca²⁺-activated ATPase. There was no correlation between the EGTA-sensitizing and the Ca²⁺-activated inhibitory activities of tropomyosin prepared under different conditions. 4. Optimum inhibition was achieved when tropomyosin and the myosin of desensitized actomyosin were present in approximately equimolar proportions. Tropomyosin had no effect on the Ca²⁺-activated ATPase of myosin measured under similar conditions. 5. Evidence is presented showing that the tropomyosin binds to desensitized actomyosin under the conditions in which the ATPase is inhibited.     

Purinergic receptor-induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium-related mechanisms

In human cervical (CaSki) cells, extracellular adenosine triphosphate (ATP) induces an acute decrease in the resistance of the lateral intercellular space (R _LIS), phase I response, followed by an increase in tight junctional resistance (R _TJ), phase II response. ATP also stimulates release of calcium from intracellular stores, followed by augmented calcium influx, and both effects have similar sensitivities to ATP (EC₅₀ of 6 μM). The objective of the study was to determine the degree to which the changes in [Ca²⁺]_i mediate the responses to ATP. 1,2-bis (2-aminophenoxy) ethane-N,N,N1,N1-tetraacetic acid (BAPTA) abrogated calcium mobilization and phase I response; in contrast, nifedipine and verapamil inhibited calcium influx and attenuated phase II response. Barium, La³⁺, and Mn²⁺ attenuated phase I response and attenuated and shortened the ionomycin-induced phase I-like decrease inR _LIS, suggesting that store depletion-activated calcium entry was inhibited. Barium and La³⁺ also inhibited the ATP-induced phase II response, but Mn²⁺ had no effect on phase II response, and in the presence of low extracellular calcium it partly restored the increase inR _TJ. KCl-induced membrane depolarization stimulated an acute decrease inR _LIS and a late increase inR _TJ similar to ATP, but only the latter was inhibited by nifedipine. KCl also induced a nifedipine-sensitive calcium influx, suggesting that acute increases in [Ca²⁺]_i, regardless of mobilization or influx, mediate phase I response. Phase II-like increases inR _TJ could be induced by treatment with diC8, and were not affected by nifedipine. Biphasic ATP-like changes inR _TE could be induced by treating the cells with ionomycin plus diC8. We conclude that calcium mobilization mediates the early decrease inR _LIS, and calcium influx via calcium channels activates protein kinase C and mediates the late increase inR _TJ.