Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent α-glucosidase

The gene for the α-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hy-drolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritimaα-glucosidase has the unusual property of requiring NAD⁺ and Mn²⁺ for activity. Co²⁺ and Ni²⁺ also activated AglA, albeit less efficiently than Mn²⁺. T. maritima AglA represents the first example of a maltodextrin-degrading α-glucosidase with NAD⁺ and Mn²⁺ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or β-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD⁺ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of d-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90°C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50°C and 70°C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h. Received: October 6, 1999 / Accepted: February 9, 2000     

Synthesis, characterization, and antibacterial and anticancer screening of {M2+–Co3+–M2+} and {Co3+–M2+} (M?is?Zn, Cd, Hg) heterometallic complexes

The cobalt(III) complexes Et₄N[Co(L¹)₂] and [Co(L²)₃] [H₂L¹ is 2,6-bis(N-(2-pyridyl)carbamoyl)pyridine and HL² is 2-(N-(2-pyridyl)carbamoyl)pyridine] were used as the building blocks for preparing a series of {M²⁺?CCo³⁺?CM²⁺} (where M?is?Zn, Cd, or Hg) and {Co³⁺?CM²⁺} (where M?is?Zn or Cd) heterometallic complexes. All heterometallic complexes were characterized using a host of spectroscopic methods (IR, NMR, and UV/vis spectroscopy and mass spectrometry), elemental analysis, and conductivity measurements. One of the representative compounds, {Hg²⁺?CCo³⁺?CHg²⁺}, was characterized crystallographically, and it was revealed that two Hg(II) ions are coordinated within the clefts created by the building block Et₄N[Co(L¹)₂]. The results of screening for anticancer activity against the human brain tumor U87 cell line and antibacterial activity against a range of resistant (Pseudomonas aeruginosa and Proteus vulgaris) as well as standard (Staphylococcus aureus SA 96, P. aeruginosa MTCC 1688, Klebsiella planticola MTCC 2272, and Escherichia coli T7) bacterial strains indicate promising activities. Notably, the observed activity was found to vary with the type of building block and the secondary metal ion present in the heterometallic complex. Treatment-induced cell death [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT and macrocolony assay), growth inhibition, cytogenetic damage, cell cycle delay, and apoptosis were studied as the parameters for cellular response.     

?-Blocker Timolol Prevents Arrhythmogenic Ca2+ Release and Normalizes Ca2+ and Zn2+ Dyshomeostasis in Hyperglycemic Rat Heart

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca²⁺ handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca²⁺ transients and reduced Ca²⁺ loading of sarcoplasmic reticulum (SR), basal intracellular free Ca²⁺ and Zn²⁺ ([Ca²⁺]_i and [Zn²⁺]_i), and spatio-temporal properties of the Ca²⁺ sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca²⁺]_i-handling regulators, such as Na⁺/Ca²⁺ exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca²⁺]_i and [Zn²⁺]_i, increased intracellular Zn²⁺ hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca²⁺ handling regulators, and prevention of Ca²⁺ leak, and thereby normalization of both [Ca²⁺]_i and [Zn²⁺]_i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.     

Extra-matrix Mg2+ limits Ca2+ uptake and modulates Ca2+ uptake–independent respiration and redox state in cardiac isolated mitochondria

Cardiac mitochondrial matrix (m) free Ca²⁺ ([Ca²⁺]_m) increases primarily by Ca²⁺ uptake through the Ca²⁺ uniporter (CU). Ca²⁺ uptake via the CU is attenuated by extra-matrix (e) Mg²⁺ ([Mg²⁺]_e). How [Ca²⁺]_m is dynamically modulated by interacting physiological levels of [Ca²⁺]_e and [Mg²⁺]_e and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg²⁺]_e modulates Ca²⁺ uptake via the CU, it also alters bioenergetics in a matrix Ca²⁺–induced and matrix Ca²⁺–independent manner. To test this, we measured changes in [Ca²⁺]_e, [Ca²⁺]_m, [Mg²⁺]_e and [Mg²⁺]_m spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl₂ (0–0.6 mM; 1 mM EGTA buffer) with/without added MgCl₂ (0–2 mM). In parallel, we assessed effects of added CaCl₂ and MgCl₂ on NADH, membrane potential (ΔΨ_m), and respiration. We found that >0.125 mM MgCl₂ significantly attenuated CU-mediated Ca²⁺ uptake and [Ca²⁺]_m. Incremental [Mg²⁺]_e did not reduce initial Ca²⁺uptake but attenuated the subsequent slower Ca²⁺ uptake, so that [Ca²⁺]_m remained unaltered over time. Adding CaCl₂ without MgCl₂ to attain a [Ca²⁺]_m from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca²⁺]_m did not additionally enhance NADH oxidation or respiration. Adding MgCl₂ did not increase [Mg²⁺]_m but it altered bioenergetics by its direct effect to decrease Ca²⁺ uptake. However, at a given [Ca²⁺]_m, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg²⁺]_e. Thus, [Mg²⁺]_e without a change in [Mg²⁺]_m can modulate bioenergetics independently of CU-mediated Ca²⁺ transport.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Cisd2 mediates lifespan: is there an interconnection among Ca2+ homeostasis,autophagy, and lifespan?

Abstract

CISD2, an evolutionarily conserved novel gene, plays a crucial role in lifespan control and human disease. Mutations in human CISD2 cause type 2 Wolfram syndrome, a rare neurodegenerative and metabolic disorder associated with a shortened lifespan. Significantly, the CISD2 gene is located within a region on human chromosome 4q where a genetic component for human longevity has been mapped through a comparative genome analysis of centenarian siblings. We created Cisd2 knockout (loss-of-function) and transgenic (gain-of-function) mice to study the role of Cisd2 in development and pathophysiology, and demonstrated that Cisd2 expression affects lifespan in mammals. In the Cisd2 knockout mice, Cisd2 deficiency shortens lifespan and drives a panel of premature aging phenotypes. Additionally, an age-dependent decrease of Cisd2 expression has been detected during normal aging in mice. Interestingly, in the Cisd2 transgenic mice, we demonstrated that a persistent level of Cisd2 expression over the different stages of life gives the mice a long-lived phenotype that is linked to an extension in healthy lifespan and a delay in age-associated diseases. At the cellular level, Cisd2 deficiency leads to mitochondrial breakdown and dysfunction accompanied by cell death with autophagic features. Recent studies revealed that Cisd2 may function as an autophagy regulator involved in the Bcl-2 mediated regulation of autophagy. Furthermore, Cisd2 regulates Ca²⁺ homeostasis and Ca²⁺ has been proposed to have an important regulatory role in autophagy. Finally, it remains to be elucidated if and how the regulation in Ca²⁺ homeostasis, autophagy and lifespan are interconnected at the molecular, cellular and organism levels.     

?-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca²⁺ and Mg²⁺ and the role of these changes in SR Ca²⁺ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N₂ to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca²⁺] <1 µM, ß-adrenergic stimulation increased luminal Ca²⁺ activation of single RyR channels, decreased luminal Mg²⁺ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg²⁺. At cytoplasmic [Ca²⁺] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg²⁺ and Ca²⁺ inhibition of RyRs. The K_a and maximum levels of cytoplasmic Ca²⁺ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca²⁺ binding to the luminal Ca²⁺ site and decreasing its affinity for luminal Mg²⁺ and 2) decreasing affinity of the low-affinity Ca²⁺/Mg²⁺ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.     

Ca2+ Sensitivity of Synaptic Vesicle Dopamine, γ-Aminobutyric Acid,and Glutamate Transport Systems

The effect of Ca2⁺ on the uptake of neurotransmitters by synaptic vesicles was investigated in a synaptic vesicle enriched fraction isolated from sheep brain cortex. We observed that dopamine uptake, which is driven at expenses of the proton concentration gradient generated across the membrane by the H⁺-ATPase activity, is strongly inhibited (70%) by 500 M Ca²⁺. Conversely, glutamate uptake, which essentially requires the electrical potential in the presence of low Cl^– concentrations, is not affected by Ca²⁺, even when the proton concentration gradient greatly contributes for the proton electrochemical gradient. These observations were checked by adding Ca²⁺ to dopamine or glutamate loaded vesicles, which promoted dopamine release, whereas glutamate remained inside the vesicles. Furthermore, similar effects were obtained by adding 150 M Zn²⁺ that, like Ca²⁺, dissipates the proton concentration gradient by exchanging with H⁺. With respect to -aminobutyric acid transport, which utilizes either the proton concentration gradient or the electrical potential as energy sources, we observed that Ca²⁺ or Zn²⁺ do not induce great alterations in the -aminobutyric acid accumulation by synaptic vesicles. These results clarify the nature of the energy source for accumulation of main neurotransmitters and suggest that stressing concentrations of Ca²⁺ or Zn²⁺ inhibit the proton concentration gradient-dependent neurotransmitter accumulation by inducing H⁺ pump uncoupling rather than by interacting with the neurotransmitter transporter molecules.     

Fluoxetine Inhibits K+ Transport Pathways (K+ Efflux, Na+-K+-2Cl− Cotransport, and Na+ Pump) Underlying Volume Regulation in Corneal Endothelial Cells

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K⁺ efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 μm by 26%, and 50 μm totally. Fluoxetine had a triphasic effect on K⁺ efflux; from 20 to 100 μm it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 μm by 25%, and 50 μm completely. Bumetanide (1 μm) inhibited RVI by 43%. In a Cl⁻-free medium, fluoxetine (50–500 μm) progressively inhibited bumetanide-insensitive K⁺ influx. The inhibitions of RVI and K⁺ influx induced by fluoxetine 20 to 50 μm were similar to those induced by 1 μm bumetanide and by Cl⁻-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K⁺ channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K⁺ efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 μm inhibits NKCC, and at 100–1000 μm inhibits the Na⁺ pump. One possible explanation for these findings is that fluoxetine could interact with K⁺-selective sites in K⁺ channels, the NKC cotransporter and the Na⁺ pump.     

��5��1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

Large conductance, calcium-activated K⁺ (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous α5β1 integrin ligand, enhances BK channel current through both Ca²⁺- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel α-subunits (mSlo) are acutely potentiated following α5β1 integrin activation. The effect occurs in a Ca²⁺-dependent manner, 1–3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca²⁺ concentrations ≥1 μm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca²⁺ sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by α5β1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel α-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca²⁺ sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.     

Relation between electron exchange and nuclear vibrations in the H 2 + +H 2 + → H 3 + +p exothermic ion-molecular reaction

The effective cross section for the H ₂ ⁺ +H ₂ ⁺ → H ₃ ⁺ +p reaction in the energy range 5.7–11.5 eV is measured by the split beam method. The maximum of the cross section at an energy of ～8 eV is related to the production of the H ₄ ⁺⁺ compound system. The reaction threshold W _thr≈5 eV provides evidence in favor of the classical model of the H ₂ ⁺ ion with the charge fixed on one of the nuclei throughout the collision event.     

Hydrochlorothiazide action on the apical Cl−, Ca2+ and K+ conductances in rabbit gallbladder epithelium. Presence of an apamin-sensitive,Ca2+-activated K+ conductance

In the rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na⁺-Cl^– symport, to depolarize the apical membrane potential and to enhance the cell-to-lumen Cl^– backflux (radiochemically measured), this increase being SITS-sensitive. To better investigate the causes of the depolarization and the Cl^– backflux increase, cells were punctured with conventional microelectrodes on the luminal side (incubation in bicarbonate-free saline at 27°C) and the apical membrane potential (V _m) was studied either with prolonged single impalements or with a set of short multiple impalements. The maximal depolarization was of 3–4 mV and was reached with 2.5 × 10^–4 m HCTZ. It was significantly enhanced by reducing luminal Cl^– concentration to 30 mm; it was abolished by SCN^–, furosemide, SITS; it was insensitive to DPC. SITS converted the depolarization into a hyperpolarization of about 4 mV; this latter was apamin, nifedipine and verapamil sensitive. It was concluded that HCTZ concomitantly opens apical Cl^– and (probably) Ca²⁺ conductances and, indirectly, a Ca²⁺-sensitive, apamin inhibitable K⁺ conductance: since the intracellular Cl^– activity is maintained above the value predicted at the electrochemical equilibrium, the opening of the apical Cl^– conductance depolarizes V _mand enhances Cl^– backflux. In the presence of apamin or verapamil, to avoid the hyperpolarizing effects due to HCTZ, the depolarization elicited by this drug was fully developed (7–10 mV) and proved to be Ca²⁺ insensitive. On this basis and measuring the transepithelial resistance and the apical/basolateral resistance ratio, the Cl^– conductance opened by HCTZ has been estimated and the Cl^– backflux increase calculated: it proved to be in the order of that observed radiochemically. The importance of this Cl^– leak to the lumen in the overall inhibition of the transepithelial NaCl transport by HCTZ has been evaluated.This research was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Rome, Italy. We are very grateful to prof. G. Meyer and dr. G. Bottà for helpful discussion and criticism.     

A permeability transition in liver mitochondria and liposomes induced by α,ω-dioic acids and Ca2+

The article examines the molecular mechanism of the Ca²⁺-dependent cyclosporin A (CsA)-insensitive permeability transition in rat liver mitochondria induced by α,ω-dioic acids. The addition of α,ω-hexadecanedioic acid (HDA) to Ca²⁺-loaded liver mitochondria was shown to induce a high-amplitude swelling of the organelles, a drop of membrane potential and the release of Ca²⁺ from the matrix, the effects being insensitive to CsA. The experiments with liposomes loaded with sulforhodamine B (SRB) revealed that, like palmitic acid (PA), HDA was able to cause permeabilization of liposomal membranes. However, the kinetics of HDA- and PA-induced release of SRB from liposomes was different, and HDA was less effective than PA in the induction of SRB release. Using the method of ultrasound interferometry, we also showed that the addition of Ca²⁺ to HDA-containing liposomes did not change the phase state of liposomal membranes—in contrast to what was observed when Ca²⁺ was added to PA-containing vesicles. It was suggested that HDA/Ca²⁺- and PA/Ca²⁺-induced permeability transition occurs by different mechanisms. Using the method of dynamic light scattering, we further revealed that the addition of Ca²⁺ to HDA-containing liposomes induced their aggregation/fusion. Apparently, these processes result in a partial release of SRB due to the formation of fusion pores. The possibility that this mechanism underlies the HDA/Ca²⁺-induced permeability transition of the mitochondrial membrane is discussed.     

Niflumic Acid Affects Store-Operated Ca2+-Permeable (SOC) and Ca2+-Dependent K+ and Cl− Ion Channels and Induces Apoptosis in K562 Cells

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl^? and Ca²⁺-activated Cl^? (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8–10 %) apoptotic effect of endoplasmic reticulum Ca²⁺ mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca²⁺-activated K⁺ (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC₅₀ for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.     

Chick brain glutamine synthetase and Mn2+−Mg2+ interactions

Glutamine synthetase (GS) from the chick brain was purified to apparent homogeneity by ammonium sulfate fractionation followed by affinity chromatography, electrofocusing and Sephadex G-150 chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate analysis in polyacrylamide gel. By sedimentation equilibrium analysis and gel electrophoresis analysis, it was shown that the enzyme has a subunit molecular weight of 45,000 and a native molecular weight of 364,000, which is consistent with an octameric structure. Sedimentation analysis in the presence of Mg²⁺ revealed three different forms of macromolecules corresponding respectively to a monomer, a tetramer and an octamer. Among eight cations tested (Ca²⁺, Co²⁺, Fe²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Zn²⁺) only Co²⁺, Mg²⁺ and Mn²⁺ supported GS activity; the order of activatory ability was Mg²⁺>Co²⁺>Mn²⁺. The maximum activating effect of Mn²⁺ occurs only within a very narrow range of concentration: with an excess of cation causing strong inhibition of GS activity. For each cation, maximal GS activity occurs at a defined cation/ATP ratio. A regulatory system in which Mn²⁺, modulates the Mg²⁺ dependent GS activity, is proposed; such cation interactions may be of significance in the intracellular control of glutamine synthesis.     

Regulation of Ca2+ Sparks by Ca2+ and Mg2+ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 microM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4-40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10(-7) in the frog or 10(-8) in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.     

Na+−Ca2+ exchange and Ca2+ channel characteristics in bovine aorta and coronary artery smooth muscle sarcolemmal membranes

Tension generation and Ca²⁺ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na⁺–Ca²⁺ exchanger and the Ca²⁺ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The K_m and V_max of Na⁺-dependent Ca²⁺ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [³H]PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher K_d and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [³H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca²⁺ channel characteristics in isolated sarcolemmal membranes.