Cytochrome c release occurs via Ca2+-dependent and Ca2+-independent mechanisms that are regulated by Bax

Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating this event remain elusive. In the present study, using isolated liver mitochondria, we demonstrate that cytochrome c release occurs via distinct mechanisms that are either Ca(2+)-dependent or Ca(2+)-independent. An increase in mitochondrial matrix Ca(2+) promotes the opening of the permeability transition (PT) pore and the release of cytochrome c, an effect that is significantly enhanced when these organelles are incubated in a reaction buffer that is based on a physiologically relevant concentration of K(+) (150 mm KCl) versus a buffer composed of mannitol/sucrose/Hepes. Moreover, low concentrations of Ca(2+) are sufficient to induce mitochondrial cytochrome c release without measurable manifestations of PT, though inhibitors of PT effectively prevent this release, indicating that the critical threshold for PT varies among mitochondria within a single population of these organelles. In contrast, Ca(2+)-independent cytochrome c release is induced by oligomeric Bax protein and occurs without mitochondrial swelling or the release of matrix proteins, although our data also indicate that Bax enhances permeability transition-induced cytochrome c release. Taken together, our results suggest that the intramitochondrial Ca(2+) concentration, as well as the reaction buffer composition, are key factors in determining the mode and amount of cytochrome c release. Finally, oligomeric Bax appears to be capable of stimulating cytochrome c release via both Ca(2+)-dependent and Ca(2+)-independent mechanisms.     

Dual mechanisms in priming of the chemoattractant-induced respiratory burst in human granulocytes. A Ca2+-dependent and a Ca2+-independent route

After interaction with so-called priming agents, the respiratory burst in human granulocytes does not become activated, but is enhanced upon subsequent stimulation with the chemoattractant FMLP. Investigating the mechanism of the priming reaction, we found that a transient rise in the cytosolic free calcium concentration [( Ca2+]i) suffices to irreversibly prime human granulocytes. Thus, platelet-activating factor (PAF) induced a transient increase in [Ca2+]i and primed the cells to an enhanced respiratory burst upon subsequent interaction with FMLP. Artificially, the transient rise in [Ca2+]i was mimicked by addition and subsequent removal of the Ca2+ ionophore ionomycin; this treatment too, primed the respiratory burst of the granulocytes. The priming induced by ionomycin was completely abolished when [Ca2+]i changes were buffered during exposure of the cells to the ionophore. The priming induced by PAF was only partially inhibited under [Ca2+]i-buffering conditions during priming, indicating that multiple pathways exist in the priming of granulocytes by PAF.     

Effects of phalloidin on K+-dependent, Ca2+-independent neurotransmitter efflux and K+-facilitated, Ca2+-dependent neurotransmitter release.

Phalloidin tightly binds to actin and converts soluble actin into depolymerization-resistant actin filaments. Phalloidin promotes the potassium-dependent, calcium-independent efflux of γ-amino butyric acid and nore-pinephrine from synaptosomes but inhibits the potassium-facilitated, calcium-dependent release of these neurotransmitters. This suggests that an actomyosin system is involved in synaptic transmission.     

Ca2+-dependent and Ca2+-independent mechanisms for phosphatidylinositol hydrolysis and 32P-labeling during cholinergic stimulation of the rat submaxillary gland in vitro

Ca²⁺ was required for carbachol-induced decreases in phosphatidylinositol (PI) and increases in phosphatidic acid (PA) concentrations during incubation of rat submaxillary gland fragments, but was not required for increases in [³²P]P_i incorporation into these phospholipids. Like carbachol, A23187 provoked a Ca²⁺-dependent decrease in PI mass. These results suggest concomitant operation of two separate mechanisms for stimulating PI hydrolysis and ³²P labeling of PA and PI during carbachol action: one mechanism is not dependent on external Ca²⁺ and is manifested by rapid labeling in a relatively small PA-PI pool; the other mechanism is dependent on Ca²⁺ and involves a large PA-PI pool which appears to have a relatively slow renewal (labeling) rate.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

Ca2+-dependent and Ca2+-independent excretion modes of salicylic acid in tobacco cell suspension culture.

14C-salicylic acid (SA) was used to monitor SA metabolism and its regulation in tobacco cell suspension culture. Two SA concentrations (20 microM and 200 microM) were used for comparison. SA was quickly taken up in both treatments, and the 200 microM-treated cells absorbed approximately 15 times that of 20 microM-treated cells within 5 min. More than 85% and 50% of the absorbed SA were excreted in free form to the culture medium within 5 h from cells treated with 200 microM and 20 microM SA, respectively. SA excretion was significantly inhibited by EGTA and the inhibition could be reversed by the addition of exogenous Ca2+ to the culture medium in the 200 microM SA treatment. However, EGTA had little or no effect on SA excretion in the 20 microM SA treatment. The data suggest that tobacco suspension-cultured cells may contain both Ca2+-dependent and Ca2+-independent pathways for SA excretion. Reduced glutathione (an active oxygen species scavenger), staurosporine (a protein kinase inhibitor), and cycloheximide (an inhibitor of de novo protein synthesis) also blocked intracellular SA excretion to the culture medium in the 200 microM but not in the 20 microM SA treatment. These data support the existence of alternative SA excretion pathways in tobacco suspension-cultured cells. Tobacco cells may use both Ca2+-dependent and Ca2+-independent excretion pathways to cope with different intracellular SA status, and the pathway influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by SA at 200 microM, but not at 20 microM.     

Angiotensin II type I receptor modulates intracellular free Mg2+ in renally derived cells via Na+-dependent Ca2+-independent mechanisms

Treatment of Madin-Darby canine kidney (MDCK) cells with the peptide hormone angiotensin II (Ang II) results in an increase in the concentrations of cytosolic free calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) with a concomitant decrease in cytosolic free Mg(2+) concentration ([Mg(2+)](i)). In the present study we demonstrate that this hormone-induced decrease in [Mg(2+)](i) is independent of [Ca(2+)](i) but dependent on extracellular Na(+). [Mg(2+)](i), [Ca(2+)](i), and [Na(+)](i) were measured in Ang II-stimulated MDCK cells by fluorescence digital imaging using the selective fluoroprobes mag-fura-2AM, fura-2AM, and sodium-binding benzofuran isophthalate (acetoxymethyl ester), respectively. Ang II decreased [Mg(2+)](i) and increased [Na(+)](i) in a dose-dependent manner. These effects were inhibited by irbesartan (selective AT(1) receptor blocker) but not by PD123319 (selective AT(2) receptor blocker). Imipramine and quinidine (putative inhibitors of the Na(+)/Mg(2+) exchanger) and removal of extracellular Na(+) abrogated Ang II-mediated [Mg(2+)](i) effects. In cells pretreated with thapsigargin (reticular Ca(2+)-ATPase inhibitor), Ang II-stimulated [Ca(2+)](i) transients were attenuated (p < 0.01), whereas agonist-induced [Mg(2+)](i) responses were unchanged. Clamping the [Ca(2+)](i) near 50 nmol/liter with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) inhibited Ang II-induced [Ca(2+)](i) increases but failed to alter Ang II-induced [Mg(2+)](i) responses. Benzamil, a selective blocker of the Na(+)/Ca(2+) exchanger, inhibited [Na(+)](i) but not [Mg(2+)](i) responses. Our data demonstrate that in MDCK cells, AT(1) receptors modulate [Mg(2+)](i) via a Na(+)-dependent Mg(2+) transporter that is not directly related to [Ca(2+)](i). These data support the notion that rapid modulation of [Mg(2+)](i) is not simply a result of Mg(2+) redistribution from intracellular buffering sites by Ca(2+) and provide evidence for the existence of a Na(+)-dependent, hormonally regulated transporter for Mg(2+) in renally derived cells.     

Demonstration of the simultaneous activation of Ca2+-independent and Ca2+-dependent ATPases from rat skeletal muscle microsomes

The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demonstrated in the presence of enough Ca2+ to provide the simultaneous activation of the (Ca2+ + Mg2+)-ATPase. It was achieved taking advantage of the delayed inorganic phosphate (Pi) release due to the formation of a phosphoenzyme complex during the Ca2+-dependent enzymatic cycle, which is evidenced in fast experiments. The microsomes were immobilized on a filter and perfused at constant flow with an incubation medium which was briefly interrupted with a pulse of appropriate reactants to activate the ATPases, at 2 degrees C. Successive samples were collected after passing through the filter, at approx. 0.1 s intervals. The Pi effluent profile coincides with the pattern of the pulse when it activates only the Ca2+-independent ATPase, it appears delayed when the pulse activates only extra Pi production by the (Ca2+ + Mg2+)-ATPase, and it includes a rapid and a delayed component when both Ca2+-independent and Ca2+-dependent ATPases are activated simultaneously by the pulse.     

Ca(2+)-dependent and Ca(2+)-independent mechanisms modulate whole-cell cationic currents in human neutrophils.

We used whole-cell, voltage-clamp methodology to study the activation and inhibition of cationic currents in neutrophil. Cationic channels involved were impermeable to N-methyl-D-glucamine and to choline, but permeable to Na+, K+, Cs+, tris(hydroxymethyl)amino-ethane, and tetraethylammonium. N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the Ca(2+)-ionophore A23187, and phorbol myristate acetate activated the cationic current. Activated currents showed voltage dependence and outward rectification. The Ca(2+)-chelator 1,2 bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate markedly inhibited A23187-induced currents, but only partially decreased phorbol ester- or chemoattractant-induced currents. Dibutyryl cAMP diminished only the chemoattractant-induced currents. The adenosine analogs 5'N-ethylcarboxamidoadenosine and N6-cyclohexyladenosine blocked the currents induced by all agents. Thus, we conclude that activation and inhibition of cationic channels in human neutrophils involve both Ca(2+)-dependent and Ca(2+)-independent mechanisms.     

电压门控钙通道钙依赖性易化和失活的分子机制

电压门控钙通道受钙依赖性易化和失活两种相互对立的反馈机制调节.不同浓度的钙离子,通过作为钙感受器的钙调蛋白的介导,主要与钙通道α1亚基羧基端的多个不连续片段发生复杂的相互作用,分别引发钙依赖性易化和失活.钙/钙调蛋白依赖性蛋白激酶Ⅱ及其它钙结合蛋白等也参与此调节过程.新近研究表明,钙通道的钙依赖性调节机制失衡与心律失常等的发病机制密切相关.     

Ca2+-dependent potentiation of muscarinic receptor-mediated Ca2+ elevation

Muscarinic receptor-mediated increases in Ca(2+) in SH-SY5Y neuroblastoma cells consist of an initial fast and transient phase followed by a sustained phase. Activation of voltage-gated Ca(2+) channels prior to muscarinic stimulation resulted in a several-fold potentiation of the fast phase. Unlike the muscarinic response under control conditions, this potentiated elevation of intracellular Ca(2+) was to a large extent dependent on extracellular Ca(2+). In potentiated cells, muscarinic stimulation also activated a rapid Mn(2+) entry. By using known organic and inorganic blockers of cation channels, this influx pathway was easily separated from the known Ca(2+) influx pathways, the store-operated pathway and the voltage-gated Ca(2+) channels. In addition to the Ca(2+) influx, both IP(3) production and Ca(2+) release were also enhanced during the potentiated response. The results suggest that a small increase in intracellular Ca(2+) amplifies the muscarinic Ca(2+) response at several stages, most notably by unravelling an apparently novel receptor-activated influx pathway.     

Ca2+-dependent gating mechanisms for dSlo, a large-conductance Ca2+-activated K+ (BK) channel.

The Ca2+-dependent gating mechanism of cloned BK channels from Drosophila (dSlo) was studied. Both a natural variant (A1/C2/E1/G3/IO) and a mutant (S942A) were expressed in Xenopus oocytes, and single-channel currents were recorded from excised patches of membrane. Stability plots were used to define stable segments of data. Unlike native BK channels from rat skeletal muscle in which increasing internal Ca2+ concentration (Cai2+) in the range of 5 to 30 microM increases mean open time, increasing Cai2+ in this range for dSlo had little effect on mean open time. However, further increases in Cai2+ to 300 or 3000 microM then typically increased dSlo mean open time. Kinetic schemes for the observed Ca2+-dependent gating kinetics of dSlo were evaluated by fitting two-dimensional dwell-time distributions using maximum likelihood techniques and by comparing observed dependency plots with those predicted by the models. Previously described kinetic schemes that largely account for the Ca2+-dependent kinetics of native BK channels from rat skeletal muscle did not adequately describe the Ca2+ dependence of dSlo. An expanded version of these schemes which, in addition to the Ca2+-activation steps, permitted a Ca2+-facilitated transition from each open state to a closed state, could approximate the Ca2+-dependent kinetics of dSlo, suggesting that Ca2+ may exert dual effects on gating.     

Translocation of protein kinase C in human polymorphonuclear neutrophils. Regulation by cytosolic Ca2(+)-independent and Ca2(+)-dependent mechanisms

[3H]Phorbol dibutyrate [( 3H]PDB) rapidly and reversibly binds to human polymorphonuclear neutrophils (PMN). Ca2+/diacylglycerol/phospholipid-dependent protein kinase C appeared to be the receptor for this binding because: a diacylglycerol, dioctanoylglycerol, competed with [3H]PDB for PMN binding sites; a blocker of protein kinase C-phospholipid interactions, sphinganine, inhibited PMN binding of [3H]PDB; and changes in cytosolic Ca2+ apparently regulated PMN binding of the label. Relevant to the last point, disrupted PMN contained 9 X 10(5) phorbol diester receptors/cell, whereas intact PMN had only 1.6 X 10(5) such receptors that were accessed by the ligand. This number fell to 1.0 X 10(5) in Ca2(+)-depleted PMN and rose to 2.5 X 10(5) in cells stimulated with the Ca2+ ionophore, ionomycin. This ionomycin effect lasted for greater than 16 min, correlated temporally with changes in cytosolic Ca2+, did not occur in Ca2(+)-depleted PMN, and was blocked by sphinganine. A second ionophore, A23187, likewise induced Ca2(+)-dependent rises in [3H]PDB binding. These results fit the standard model, wherein rises in cytosolic Ca2+ cause protein kinase C to translocate from cytosol to plasmalemma and thereby become more available to [3H]PDB. In contrast, two humoral agonists, N-formyl-Met-Leu-Phe (fMLP) and leukotriene (LT)B4, had actions that did not fit this model. They stimulated PMN to increase the availability of PDB binding sites by a sphinganine-sensitive mechanism, but their actions differed from those of ionophores. They induced biphasic (t = 15 and 60 s) increases in [3H]PDB binding while eliciting monophasic (t = 15 s), short-lived (t less than 1 min) rises in cytosolic Ca2+. In Ca2(+)-depleted PMN, moreover, fMLP and LTB4 stimulated slow (t greater than or equal to 30 s), monophasic, prominent rises in [3H]PDB binding and binding site number without appreciably altering cytosolic Ca2+. We suggest, therefore, that fMLP and LTB4 translocate protein kinase C using two sequential mechanisms. The first involves Ca2+ transients and thus produces abrupt (t = 15 s), rapidly reversing responses. The second mechanism uses an unrelated signal to effect a more slowly evolving (t = 60 s) movement of protein kinase C to plasmalemma. Hence, the standard model does not explain all instances of protein kinase C translocation, and a cytosolic Ca2(+)-independent signal contributes to the regulation of protein kinase C as well as those responses elicited by the effector enzyme.     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

Ca2+-binding and Ca2+-independent respiratory NADH and NADPH dehydrogenases of Arabidopsis thaliana

Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca(2+) binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca(2+), and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca(2+) binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca(2+) stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca(2+)-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca(2+). The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.     

Ca2+-dependent K+ transport in lymphocytes

The treatment of rat thymocytes with A23187 + Ca2+, ascorbate-phenazine methosulphate or propranolol induced quinine-sensitive fluxes of K+ (Rb+) suggesting the presence in the cell membrane of Ca2+-dependent K+ channels. Concanavalin A induced K+ channel activation only at very high doses (13 micrograms/ml). Neither quinine nor the increase of the K+ concentration in the medium to 30 mM prevented the stimulation of amino acid transport induced by concanavalin A, suggesting that the Ca2+-dependent K+ channel is not involved in the early phenomena of lymphocyte activation.