microM Ca2 Research Articles

Calcium balance and mechanotransduction in rat cochlear hair cells.

Auditory transduction occurs by opening of Ca(2+)-permeable mechanotransducer (MT) channels in hair cell stereociliary bundles. Ca(2+) clearance from bundles was followed in rat outer hair cells (OHCs) using fast imaging of fluorescent indicators. Bundle deflection caused a rapid rise in Ca(2+) that decayed after the stimulus, with a time constant of about 50 ms. The time constant was increased by blocking Ca(2+) uptake into the subcuticular plate mitochondria or by inhibiting the hair bundle plasma membrane Ca(2+) ATPase (PMCA) pump. Such manipulations raised intracellular Ca(2+) and desensitized the MT channels. Measurement of the electrogenic PMCA pump current, which saturated at 18 pA with increasing Ca(2+) loads, indicated a maximum Ca(2+) extrusion rate of 3.7 fmol x s(-1). The amplitude of the Ca(2+) transient decreased in proportion to the Ca(2+) concentration bathing the bundle and in artificial endolymph (160 mM K(+), 20 microM Ca(2+)), Ca(2+) carried 0.2% of the MT current. Nevertheless, MT currents in endolymph displayed fast adaptation with a submillisecond time constant. In endolymph, roughly 40% of the MT current was activated at rest when using 1 mM intracellular BAPTA compared with 12% with 1 mM EGTA, which enabled estimation of the in vivo Ca(2+) load as 3 pA at rest. The results were reproduced by a model of hair bundle Ca(2+) diffusion, showing that the measured PMCA pump density could handle Ca(2+) loads incurred from resting and maximal MT currents in endolymph. The model also indicated the endogenous mobile buffer was equivalent to 1 mM BAPTA.

Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride: STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca(2+)-ATPase E1PCa(2).Mg, a complex of E1Ca(2).BeF(x), was successfully developed by addition of beryllium fluoride and Mg(2+) to the Ca(2+)-bound state, E1Ca(2). In E1Ca(2).BeF(x), most probably E1Ca(2).BeF(3)(-), two Ca(2+) are occluded at high affinity transport sites, its formation required Mg(2+) binding at the catalytic site, and ADP decomposed it to E1Ca(2), as in E1PCa(2).Mg. Organization of cytoplasmic domains in E1Ca(2).BeF(x) was revealed to be intermediate between those in E1Ca(2).AlF(4)(-) ADP (transition state of E1PCa(2) formation) and E2.BeF(3)(-).(ADP-insensitive phosphorylated intermediate E2P.Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca(2).BeF(x) in contrast to no superfluorescence of TNP-AMP bound to E1Ca(2).AlF(x). E1Ca(2).BeF(x) with bound TNP-AMP slowly decayed to E1Ca(2), being distinct from the superfluorescent complex of TNP-AMP with E2.BeF(3)(-), which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca(2).BeF(x) mimics E1PCa(2).Mg, and between those of E1Ca(2).AlF(4)(-).ADP and E2.BeF(3)(-). E1Ca(2).BeF(x) at low 50-100 microm Ca(2+) was converted slowly to E2.BeF(3)(-) releasing Ca(2+), mimicking E1PCa(2).Mg --> E2P.Mg + 2Ca(2+). Ca(2+) replacement of Mg(2+) at the catalytic site at approximately millimolar high Ca(2+) decomposed E1Ca(2).BeF(x) to E1Ca(2). Notably, E1Ca(2).BeF(x) was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca(2+) with 15 mm Mg(2+). Also, stable E1Ca(2).BeF(x) was produced from E2.BeF(3)(-) at 0.7 mm lumenal Ca(2+) by binding two Ca(2+) to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P. Mg + 2Ca(2+) --> E1PCa(2).Mg.

Heterogeneity in function of small artery smooth muscle BKCa: involvement of the β1‐subunit

Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage-gated Ca2+ channels. To prevent positive feedback and excessive pressure-induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca2+-activated K+ channels (BKCa) provides an opposing hyperpolarizing influence reducing Ca2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K+ currents were determined at pipette [Ca2+] of 100 nM or 5 microM in the presence and absence of the BKCa inhibitor, iberiotoxin (IBTX; 0.1 microM). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca2+ levels. At 5 microM Ca2+, cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 +/- 1.9 vs 45.5 +/- 1.7 pA pF(-1) at +70 mV). Studies with IBTX suggested that the differences in K+ conductance at 5 microM intracellular [Ca2+] were largely due to activity of BKCa. 17beta-Oestradiol (1 microM), reported to potentiate BKCa current via the channel's beta-subunit, caused a greater effect on whole cell K+ currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the alpha-subunit-selective BKCa opener, NS-1619 (20 microM), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the beta1 subunit and a decrease in the beta1:alpha ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BKCa protein and the beta1:alpha-subunit ratio. The data support vascular heterogeneity with respect to the activity of BKCa in terms of both beta-subunit regulation and interaction with SR-mediated Ca2+ signalling.

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca(2+) uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca(2+) transport system (Bazhenova et al. J Biol Chem 273:4372-4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96-100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352-1356, 2000; Deryabina et al. J Biol Chem 276:47801-47806, 2001) were very resistant to Ca(2+) overload. However, exposure of yeast mitochondria to 50-100 microM Ca(2+) in the presence of the Ca(2+) ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca(2+)/nH(+)-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca(2+)- ETH129-induced activation of the Ca(2+)/H(+)-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca(2+) overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319-331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37-51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca(2+) uptake and is differently regulated compared to the mPTP of animal mitochondria.

Acrosome content release in streptolysin O permeabilized mouse spermatozoa.

Sperm cell plasma membrane and the outer acrosomal membrane fuse profusely during the acrosome reaction. The process is triggered by extracellular signals that elicit several intracellular events leading ultimately to membrane fusion. We have developed a streptolysin O permeabilizing protocol that selectively affects the spermatozoon plasma membrane without causing a significant loss of the acrosomal content. Most of the acrosomal acid phosphatase remained sperm-associated even after a 20 min incubation at 37 degrees C. However, the presence of 100 microM Ca2+ in the incubation buffer stimulates the release of the enzyme. The reaction was followed biochemically, measuring the acid phosphatase activity released to the medium and morphologically by the binding of fluorescein isothiocynate-conjugated peanut agglutinin and by electron microscopy. The results show that the streptolysin O permeabilized spermatozoon is a promising model for studying the complex set of events mediating and regulating the acrosome reaction.

Fluorescent Recognition of Potassium and Calcium Ions Using Functionalised CdSe / ZnS Quantum Dots

Schiff base receptor 1a has been synthesised and attached to the surface of preformed CdSe/ZnS Quantum Dots (QDs) to form QD-conjugate 2a. While 1a was determined to be selective for Mg2+, 2a demonstrated selectivity for both K+ and Ca2+ when tested against a range of physiologically and environmentally relevant cations by changes in the fluorescence spectra. Thus, the nanoparticle surface functions as a scaffold for the organisation of receptors enabling semi-selective binding. The fluorescence response was shown to be linear between 15-50 microM for K+ and 2-35 microM for Ca2+. It was also demonstrated that 2a could measure both K+ and / or Ca2+ in solutions containing both ions.

Structural basis for inhibition of mammalian adenylyl cyclase by calcium.

Type V and VI mammalian adenylyl cyclases (AC5, AC6) are inhibited by Ca(2+) at both sub- and supramicromolar concentration. This inhibition may provide feedback in situations where cAMP promotes opening of Ca(2+) channels, allowing fine control of cardiac contraction and rhythmicity in cardiac tissue where AC5 and AC6 predominate. Ca(2+) inhibits the soluble AC core composed of the C1 domain of AC5 (VC1) and the C2 domain of AC2 (IIC2). As observed for holo-AC5, inhibition is biphasic, showing "high-affinity" (K(i) = approximately 0.4 microM) and "low-affinity" (K(i) = approximately 100 microM) modes of inhibition. At micromolar concentration, Ca(2+) inhibition is nonexclusive with respect to pyrophosphate (PP(i)), a noncompetitive inhibitor with respect to ATP, but at >100 microM Ca(2+), inhibition appears to be exclusive with respect to PP(i). The 3.0 A resolution structure of Galphas.GTPgammaS/forskolin-activated VC1:IIC2 crystals soaked in the presence of ATPalphaS and 8 microM free Ca(2+) contains a single, loosely coordinated metal ion. ATP soaked into VC1:IIC2 crystals in the presence of 1.5 mM Ca(2+) is not cyclized, and two calcium ions are observed in the 2.9 A resolution structure of the complex. In both of the latter complexes VC1:IIC2 adopts the "open", catalytically inactive conformation characteristic of the apoenzyme, in contrast to the "closed", active conformation seen in the presence of ATP analogues and Mg(2+) or Mn(2+). Structures of the pyrophosphate (PP(i)) complex with 10 mM Mg(2+) (2.8 A) or 2 mM Ca(2+) (2.7 A) also adopt the open conformation, indicating that the closed to open transition occurs after cAMP release. In the latter complexes, Ca(2+) and Mg(2+) bind only to the high-affinity "B" metal site associated with substrate/product stabilization. Ca(2+) thus stabilizes the inactive conformation in both ATP- and PP(i)-bound states.

In vitro modulation of the cardiac ryanodine receptor activity by Homer1

The Homer protein family allows clustering and/or functional modulation of many proteins from different calcium signalling complexes including those formed by the ryanodine receptor (RyR) Ca(2+) release channel in skeletal muscle and the heart. Homer1b/c and the cardiac RyR (RyR2) are strongly expressed in the heart and neurons where their interaction with each other may modulate Ca(2+) signalling. However, functional interactions between Homer1b and RyR2 have been poorly defined. Our preliminary data and similar consensus binding sites for Homer in RyR2 and skeletal RyR (RyR1) proteins, led to the hypothesis that Homer may similarly regulate both RyR isoforms. Single-channel and [(3)H]ryanodine binding data showed that RyR2 and RyR1 activity increased to a maximum with ~50-100 nM Homer1b and fell with Homer1b > 200 nM. Homer1b (50 nM) activated RyR2 and RyR1 at all cytosolic [Ca(2+)]; estimated EC(50) value of RyR2 diminished from ~2.8 microM Ca(2+) (control) to ~1.9 microM Ca(2+) in the presence of 50 nM Homer1b. Short Homer1 (lacking the coiled-coil multimerisation domain) and Homer1b similarly modulated RyR2, indicating an action through ligand binding, not mutimerisation. These actions of Homer were generally similar in RyR2 and RyR1. The strong functional interactions suggest that Homer1 is likely to be an endogenous modulator of RyR channels in the heart and neurons as well as in skeletal muscle.

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca(2+) level ([Ca(2+)](i)), and Ca(2+) sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 microM) inhibited high K(+) (25 mM and 40 mM)- or histamine (3 microM)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K(+)-evoked contractions. Spontaneous changes in [Ca(2+)](i) and contractions were inhibited by forskolin (1 microM) without changing the resting [Ca(2+)](i). Forskoln (10 microM) inhibited muscle tension more strongly than [Ca(2+)](i) stimulated by high K(+), and thus shifted the [Ca(2+)](i)-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 microM) inhibited both [Ca(2+)](i) and muscle tension without changing the [Ca(2+)](i)-tension relationship. In alpha-toxin-permeabilized tissues, forskolin (10 microM) inhibited the 0.3 microM Ca(2+)-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca(2+)-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca(2+) sensitivity of contractile elements in high K(+)-stimulated muscle and a decrease in [Ca(2+)](i) in histamine-stimulated muscle.

Abstract 1783: Pro-Apoptotic Bnip3 Mediates Permeabilization of Mitochondria and Release of Cytocrome c via a Novel Mechanism

Bnip3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 proteins and is associated with mitochondrial dysfunction and cell death in the myocardium. The pro-apoptotic Bcl-2 proteins mediate mitochondrial dysfunction independent of the mitochondrial permeability transition pore (mPTP). However, Bnip3 has been reported to mediate cell death via the mPTP. In this study, we investigated the mechanism(s) by which Bnip3 causes mitochondrial dysfunction. Using a mitochondrial swelling assay to assess pore opening, we found that addition of 200 microM Ca2+ to mitochondria isolated from rat hearts induced rapid swelling of mitochondria and release of cytochrome c (cyto c). Bnip3 also induced mitochondrial swelling and cyto c release, but always at a slower rate and to a greater degree, suggesting that Bnip3 mediates swelling via a different mechanism. Cyclosporin A (CsA), an inhibitor of mPTP opening, prevented Ca2+-induced swelling and cyto c release, but had no effect on Bnip3. Another BH3-only protein, tBid, caused release of cyto c but failed to induce swelling of mitochondria. Interestingly, Bnip3, but not Ca2+ and tBid, induced release of the matrix protein MnSOD. Cyclophilin D (cycD) is an essential component of the mPTP and heart mitochondria isolated from cycD−/− mice were resistant to Ca2+-, but not to Bnip3-induced swelling and cyto c release. Also, tBid caused cyto c release without mitochondrial swelling in the absence of cycD. To further explore the mPTP as a downstream effector of Bnip3-mediated cell death, we assessed cell death in mouse embryonic fibroblasts (MEFs) isolated from wild type (wt) and cycD−/− mice. Infection with an adenovirus expressing Bnip3 caused significant cell death in wt (52.8±1.8%) and cycD−/− (61.8±6.1%) MEFs as measured by LDH release. In addition, both Bnip3 and opening of the mPTP have been reported to initiate upregulation of autophagy. Monitoring of GFP-LC3 incorporation into autophagosomes by fluorescence microscopy revealed that Bnip3 infection induced autophagy in wt (86.5±6.6%) and cycD−/− (96.4±1.4%) MEFs (n=3, p&lt;0.05). Thus, these studies suggest that Bnip3 mediates permeabilization of the inner and outer mitochondrial membranes via a novel mechanism that is different from other BH3-only proteins. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Physicochemical and functional characterization of the polymerization process of the Geodia cydonium lectin.

The extracellularly localized, galactose-specific lectin from the sponge Geodia cydonium binds at one class of sites, 40 mol Ca2+/mol lectin with an association constant (Ka) of 0.3 X 10(6)M-1. Stoichiometric calculations reveal that in the extracellular milieu 22 mol Ca2+ (maximum) are complexed per mol lectin. Binding of Ca2+ to the lectin increases its apparent Mr from 44000 to 56000 (electrophoretic determination) or from 36500 to 53500 (high-pressure liquid gel chromatographical determination); the s20, w increases from 4.3 S to 4.5 S if Ca2+ is added to the lectin. In the presence of Ca2+ the lectin undergoes a conformational change perhaps by expanding the carbohydrate side chains which are terminated by galactose. Subsequently the lectin molecules polymerize to large three-dimensional clumps (diameter up to 8 micron). Turbidimetric studies reveal an inhibition of the lectin polymerization by lactose. The Ka of the lectin-lectin polymerization rises from 0.9 X 10(6)M-1 to 14.0 X 10(6)M-1 after increasing the Ca2+ concentration (from 1 microM to 100 microM). Parallel with this increase in affinity, the Ka value of the lectin-aggregation factor binding drops from 41.2 X 10(6)M-1 (1 microM Ca2+) to 1.3 X 10(6)M-1 (100 microM Ca2+). In the absence of Ca2+, the Geodia lectin forms 1-10-micron two-dimensional sheets in the presence of homologous glycoconjugates. Cell binding experiments with polyacrylamide gels, containing covalently bound galactose, show that both homologous (Geodia cydonium) and heterologous cells (L5178y) bind with a higher affinity to the lectin-polymer matrix than to the lectin-monomer one. These data suggest that lectin-polymer structures, together with lectin-glycoconjugate associates, are components of the cell-substrate adhesion system(s) of sponges in vivo.

Interaction of Group IA Phospholipase A2 with Metal Ions and Phospholipid Vesicles Probed with Deuterium Exchange Mass Spectrometry

Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A 2 (GIA PLA 2) was carried out in the presence of metal ions Ca (2+) and Ba (2+) and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein-lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large alpha-helices of residues 43-53 and 89-101, which suggests that these alpha-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca (2+) or Ba (2+) ions decreased the deuterium exchange rates for five regions of the protein (residues 24-27, 29-40, 43-53, 103-110, and 111-114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24-27 and 103-110 which showed greater changes for Ca (2+). The crystal structure of the N. naja naja GIA PLA 2 contains a single Ca (2+) bound in the catalytic site, but the crystal structures of related PLA 2s contain a second Ca (2+) binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA 2 does in fact bind two Ca (2+) ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 microM Ca (2+) present at 0 degrees C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3-8, 18-21, and 56-64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate.

Desflurane Affords Greater Protection Than Halothane in the Function of Mitochondria Against Forebrain Ischemia Reperfusion Injury in Rats

Halothane and desflurane have been shown to attenuate neuronal injury; however, the effects of these anesthetics on mitochondria are unclear. We investigated whether halothane and desflurane affect the function of mitochondria after cerebral ischemia in rats. Forty male Wistar rats were randomly divided into four groups (n = 10 each): sham group; 1.5 minimal alveolar concentration (MAC) halothane group; 1.0 MAC desflurane; and 1.5 MAC desflurane group. Forebrain ischemia was induced after 40-min inhalation of 1.5 MAC halothane, 1.0 MAC or 1.5 MAC desflurane by clamping the bilateral common carotid arteries and decreasing arterial blood pressure. After isolation of the brain mitochondria, mitochondrial membrane permeability was assayed spectrophotometrically with 40-200 microM Ca(2+), and mitochondrial membrane potentials were measured by a fluorospectrophotometer with the addition of rhodamine 123. The activities of mitochondrial respiratory chain complexes were also assayed spectrophotometrically. The results showed obvious mitochondrial swelling, loss of membrane potential with the addition of Ca(2+), and inhibition of the activities of complexes I + III and IV after forebrain ischemia reperfusion injury. Compared with the 1.5 MAC halothane group, 1.0 and 1.5 MAC desflurane reduced mitochondrial swelling by 23.9% (P < 0.001) and 23.2% (P < 0.001), whereas membrane potential dissipation was suppressed by 22.4% (P = 0.013) and 20.4% (P = 0.027). The activities of complexes I + III and IV were better preserved in 1.0 MAC and 1.5 MAC desflurane groups than in the 1.5 MAC halothane group by 34.6% (P = 0.027), 38.7% (P = 0.011), 53.9% (P = 0.009), and 55.8% (P = 0.007), respectively. Desflurane shows better preservation of mitochondrial function at 4 h after cerebral ischemia reperfusion injury, indicated by inhibition of mitochondrial swelling, increase of membrane potential, and improvement of functions of mitochondria respiratory complexes I + III and IV when compared with halothane.

Changes in calcium‐dependent membrane permeability properties in mitochondria of livers from arthritic rats

The involvement of the mitochondrial permeability transition pore (PTP) in the responses of mitochondria from adjuvant-induced arthritic rats to Ca(2+) addition was investigated. The respiratory activity, the Ca(2+)-induced osmotic swelling and the electrophoretic (45)Ca(2+) uptake were evaluated in the absence and in the presence of cyclosporin A (CsA), a well-known inhibitor of the mitochondrial PTP. The Ca(2+)-induced mitochondrial permeability transition (MPT) process occurred in mitochondria from arthritic rats even in the presence of a low Ca(2+) concentration. Whereas in the normal condition, the Ca(2+)-induced uncoupling of oxidative phosphorylation and osmotic swelling was observed in the presence of 10 or 20 microM Ca(2+) concentration, in the arthritic condition, these events occurred at 1.0 microM concentration. In addition, mitochondria from arthritic rats presented an impaired ability to accumulate (45)Ca(2+). All these effects were completely prevented by the administration of CsA. The results of the present study suggest that the higher sensitivity of mitochondria from arthritic rats to Ca(2+)-induced MPT may be an important factor in the pathogenesis of the arthritis disease.

A Plant Ca2+ Pump, ACA2, Relieves Salt Hypersensitivity in Yeast: MODULATION OF CYTOSOLIC CALCIUM SIGNATURE AND ACTIVATION OF ADAPTIVE Na+HOMEOSTASIS

Stress responses in both plants and yeast utilize calcium-mediated signaling. A yeast strain, K616, which lacks Ca(2+) pumps, requires micromolar Ca(2+) for growth. In medium containing 100 microM Ca(2+), K616 can withstand osmotic stress (750 mM sorbitol) and ionic stress (300 mM KCl) but not hypersodic stress (300 mM NaCl). Heterologous expression of the endoplasmic reticulum-located Arabidopsis thaliana Ca(2+)-ATPase, ACA2, permits K616 to grow under NaCl stress even in Ca(2+)-depleted medium. All stresses tested generated transient elevation of cytosolic Ca(2+) in wild type yeast, K601, whereas NaCl alone induced prolonged elevation of cytosolic Ca(2+) in K616. Both the Ca(2+) transient and survival of cultures subjected to NaCl stress was similar for the ACA2 transformant and K601. However, whereas K601 maintained low cytosolic Na(+) predominantly by pumping it out across the plasma membrane, the transformant sequestered Na(+) in internal organelles. This sequestration requires the presence of an endomembrane Na(+)/H(+)-antiporter, NHX1, which does not play a significant role in salt tolerance of wild type yeast except at acidic pH. Transcript levels of the plasma membrane Na(+)-ATPase, ENA1, were strongly induced only in K601, whereas NHX1 was strongly induced in both K601 and the ACA2 transformant. The calmodulin kinase inhibitor KN62 significantly reduced the salt tolerance of the ACA2 transformant and the transcriptional induction of NHX1. Thus, the heterologous expression of a plant endomembrane Ca(2+) pump results in the rapid depletion of cytosolic Ca(2+) and the activation of an alternate mechanism for surviving saline stress.

Effects of perfluorooctane sulfonate (PFOS) on swimming behavior and membrane potential of paramecium caudatum

Persistent perfluorinated organic compounds such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were distributed widely in the global. PFOS (15 microM or higher) caused backward swimming of paramecia. The Triton-extracted paramecia, where the membrane was disrupted and the externally applied chemicals are freely accessible to the ciliary apparatus, showed forward swimming up to 0.1 microM Ca2+ in the medium and backward swimming at about 0.2 microM and higher. PFOS (0.1 mM) did not change the relationship between the swimming directions and free Ca2+ concentrations. Effects of various surfactants including PFOS and PFOA on the swimming direction of paramecia were compared with the hemolysis of mouse erythrocytes as an indicator of surfactant activities. The hemolysis did not correlate with their swimming behavior. PFOS caused triphasic membrane potential changes both in the wild-type paramecia and caudatum non-reversal (CNR) mutants, the latter is defective in voltage-gated Ca2+ channels. An action potential of the wild-type specimen was induced at lower current intensity when PFOS was present in the medium. Voltage-clamp study indicated that PFOS had no effect on the depolarization-induced Ca2+ influx responsible for the action potential. The membrane potential responses obtained were similar to those obtained by the application of some bitter substances such as quinine that activate chemoreceptors of paramecia. Since the CNR specimens did not exhibit PFOS-induced backward swimming at concentrations examined, the backward swimming is attributable to the influx of Ca2+ into the cilia through voltage-gated Ca2+ channels. The Ca2+ channels are most probably activated by the depolarizing receptor potentials resulted from the PFOS-induced activation of chemoreceptors.

Characterization of a Tobacco TPK-type K+ Channel as a Novel Tonoplast K+ Channel Using Yeast Tonoplasts

The tonoplast K(+) membrane transport system plays a crucial role in maintaining K(+) homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K(+) channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased approximately 2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K(+) over Na(+). NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca(2+), but a cytosolic concentration of 45 microM Ca(2+) resulted in a 2-fold increase in the amplitude of the K(+) current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K(+) currents. These results show that NtTPK1 is a novel tonoplast K(+) channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.

Effect of Chemical or Electrical Activation of Bovine Oocytes on Blastocyst Development and Quality

Activation of in vitro-matured (IVM) oocytes is essential for successful embryo production following nuclear transfer (NT) or intracytoplasmic sperm injection (ICSI). This study was designed to compare the rates of blastocyst production and embryo quality (as measured by numbers of viable cells) following parthenogenetic activation with electrical pulse or the use of two different calcium ionophores, A23187 (CA) or ionomycin (IO), with or without the addition of bovine serum albumin (BSA). IVM oocytes with a first polar body were randomly allocated to five treatment groups: CA (5 microM CA, 5 min; n = 88), CA + BSA (5 microM CA, 5 min; BSA, 5 min; n = 90), IO (5 microM IO, 5 min; n = 91), IO + BSA (5 microM IO, 5 min; BSA, 5 min; n = 86) and EL (two pulses of 1.5 kV/cm, 20 micros; n = 120). Blastocyst rates were higher (p < 0.05) for CA (54.4%), IO (51.4%) and EL (54.5%) than for IO + BSA (18.3%). Treatment CA + BSA (39.8%) did not differ from the others. There was no difference (p > 0.05) among treatments in total number of cells. However, the percentage of viable cells was reduced in CA (49.9%), CA + BSA (45.8%), IO (64.9%), IO + BSA (50.9%) compared with EL (82.7%). In summary, the addition of BSA to the IO treatment had an adverse effect on blastocyst production rates. Although there was no difference between electrical stimulation and chemical activation on blastocyst production rates, electrical activation resulted in blastocysts with a higher percentage of viable cells.

Novel substituted 1,4-anthracenediones with antitumor activity directly induce permeability transition in isolated mitochondria

Synthetic analogs of 1,4-anthraquinone (AQ code number), which block nucleoside transport, decrease DNA, RNA and protein syntheses, trigger cytochrome c release without caspase activation, induce apoptotic DNA fragmentation and inhibit the proliferation of wild-type and multidrug resistant tumor cells in the nM range in vitro, rapidly cause the collapse of mitochondrial transmembrane potential in cell and cell-free systems. Because mitochondrial permeability transition (MPT) requires more than depolarization to occur, antitumor AQs were tested for their ability to directly trigger specific markers of MPT in isolated mitochondria. In contrast to a spectrum of conventional anticancer drugs that are inactive, various AQs interact with isolated mitochondria in a concentration- and time-dependent manner to rapidly cause large amplitude swelling and Ca2+ release in relation with their effectiveness against L1210, HL-60 and LL/2 tumor cells in vitro. Indeed, the lead antitumor AQ8, AQ9 and AQ17 are also the most effective inducers of MPT in isolated mitochondria, whereas all AQ derivatives devoid of anti-proliferative activity also fail to trigger mitochondrial swelling and Ca2+ release. Moreover, the ability of 4 microM AQ17 to maximally induce mitochondrial swelling and Ca2+ release within 15 min is similar to that of classic MPT-inducing agents, such as 5 microg/ml alamethicin, 200 microM atractyloside, 5 microM phenylarsine oxide, 100 microM arsenic trioxide and a 100 microM Ca2+ overload. Interestingly, AQ17 requires a priming concentration of 20 microM Ca2+ to trigger mitochondrial swelling and Ca2+ release and these 0.1 microM ruthenium red-sensitive MPT events are abolished by 1 microM cyclosporin A, 2 mM ADP and 20 microM bongkrekic acid, which block components of the permeability transition pore (PTP), and also inhibited by 50-100 microM of various ubiquinones, which interact with the quinone binding site of the PTP and raise the Ca2+ load required for PTP opening. Hence, antitumor AQs that target isolated mitochondria and trigger MPT might directly interact with components of the PTP to induce conformational changes that increase its Ca2+ sensitivity and transition from the closed to the open state.

Activation of Large-Conductance Calcium-Activated Potassium Channels by Puerarin: The Underlying Mechanism of Puerarin-Mediated Vasodilation

Puerarin is the main isoflavone found in Pueraria lobata (Willd) Ohwi, which has been used in therapy for various cardiovascular diseases. The present study examined the effects of puerarin on the large-conductance voltage- and Ca2+-activated potassium (BK(Ca)) channel and on rat thoracic aortas. BK(Ca) channels encoded with either alpha (BK-alpha) or alpha/beta subunits (BK-alpha+beta1) were heterologously expressed in Xenopus oocytes or human embryonic kidney 293 cells. The activities of BK(Ca) channels were measured using excised patch-clamp recordings. Puerarin activated BK-alpha+beta1 currents with a half-maximal concentration (EC50) of 0.8 nM and a Hill coefficient of 1.11 at 10 microM Ca2+ and with an EC50 of 12.6 nM and a Hill coefficient of 1.08 at 0 microM Ca2+. Puerarin (1 nM) induced a 16-mV leftward shift in the conductance-voltage curve for BK-alpha+beta1 currents at 10 microM Ca2+ and at 100 nM induced a 26-mV leftward shift at 0 microM Ca2+. Puerarin mainly increased the BK-alpha+beta1 channel open probability without changing the unitary conductance. Activation was also detected in the absence of the beta1 subunit. A deglycosylated analog of puerarin, daidzein, also activated BK(Ca) channels with weaker potency. In addition, puerarin (0.1 to 1000 microM) caused concentration-dependent relaxations of rat thoracic aortic rings contracted with 1 microM noradrenaline bitartrate (EC50 = 1.1 microM). These were significantly inhibited by 50 nM iberiotoxin, a specific blocker of BK(Ca) channels. This is the first study demonstrating that puerarin activates BK(Ca) channels, especially BK-alpha+beta1 channels. The activation of the BK(Ca) channel probably contributes to the puerarin-mediated vasodilation action.