The connection between ionophore-mediated Ca2+-movements and intermediary metabolism in human red cells. II. Site and mode of glycolytic activation during Ca2+-loading

Human red cells (RBC) respond to moderate Ca²⁺-loading with increased ATP consumption and stimulation of glycolytic flux. 1. Ca²⁺-induced metabolite transitions at different pH-values showed a clearcut crossover at the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase ($\text{GAPDH}\text{PGK}\text{)-steps}$. 2. The behavior of glycolytic metabolites in iodoacetate-treated, GAPDH-inhibited, and in phosphoenolpyruvate-loaded RBC ruled out activation of hexokinase, phosphofructokinase and pyruvate kinase. 3. Glycolytic stimulation is linked to Ca²⁺-extrusion rate and not to the loaded Ca²⁺. 4. Adenine nucleotides and inorganic phosphate could be ruled out as the connecting link between glycolytic activation and Ca²⁺-extrusion. 5. NADH oxidation was observed at all pH-values studied when the RBC were incubated either at low or high extracellular potassium. NADH is product-inhibitor of GAPDH. The concentration (34 μM) of thermodynamically free NADH calculated from the $\text{GAPDH}\text{PGK}$ equilibrium reactants was in the inhibitory range: any decrease in NADH is therefore followed by activation of GAPDH. $\text{NAD}\text{NADH}$ ratio seems to be the connecting link between ATP consuming ion transport and ATP generation by glycolysis.     

The connection between ionophore-mediated Ca-movements and intermediary metabolism in human red cells. I. Relationships between Ca-loading, ATP-consumption and glycolytic flux

Human red cells (RBC) were loaded with moderate amounts of Ca²⁺ by the ionophore A23187. Quantitative relationships between Ca²⁺-loading, ATP consumption and glycolytic flux were established. 1. Ca²⁺-loading is accompanied by ATP depletion. A maximum ATP consumption of approximately 10 mmoles/l RBC/h was estimated. 2. There is a positive correlation between lactate formation and Ca²⁺-loading. This is linear from 1.4 to about 4 mmoles lactate/l RBC/h. 3. Ca²⁺-induced glycolytic stimulation seems not to be mediated by adenine nucleotides. A wide range of energy charges and very different adenine nucleotide patterns were associated with the same stimulation of lactate production. 4. The turnover of the (Ca²⁺-Mg²⁺)-ATPase and its share in the Ca²⁺-stimulated ATP consuming processes were estimated with inhibitors. 1 mM La³⁺ inhibited both Ca²⁺-outward transport and ATP consumption by 80%. The remaining 20% of the ATP consumption was accounted for by the (Na⁺-K⁺)-ATPase. 5. A Ca²⁺ extrusion to ATP consumption molar ratio of 2:1 was found. However, when ATP consumption was due to the breakdown of previously accumulated glycolytic intermediates, the ratio dropped to about 1.     

The connection between ionophore-mediated Ca2+-movements and intermediary metabolism in human red cells. I. Relationships between Ca2+-loading,ATP-consumption and glycolytic flux

Human red cells (RBC) were loaded with moderate amounts of Ca²⁺ by the ionophore A23187. Quantitative relationships between Ca²⁺-loading, ATP consumption and glycolytic flux were established. 1. Ca²⁺-loading is accompanied by ATP depletion. A maximum ATP consumption of approximately 10 mmoles/l RBC/h was estimated. 2. There is a positive correlation between lactate formation and Ca²⁺-loading. This is linear from 1.4 to about 4 mmoles lactate/l RBC/h. 3. Ca²⁺-induced glycolytic stimulation seems not to be mediated by adenine nucleotides. A wide range of energy charges and very different adenine nucleotide patterns were associated with the same stimulation of lactate production. 4. The turnover of the (Ca²⁺-Mg²⁺)-ATPase and its share in the Ca²⁺-stimulated ATP consuming processes were estimated with inhibitors. 1 mM La³⁺ inhibited both Ca²⁺-outward transport and ATP consumption by 80%. The remaining 20% of the ATP consumption was accounted for by the (Na⁺-K⁺)-ATPase. 5. A Ca²⁺ extrusion to ATP consumption molar ratio of 2:1 was found. However, when ATP consumption was due to the breakdown of previously accumulated glycolytic intermediates, the ratio dropped to about 1.     

Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells

Ca²⁺-transport and its energy consumption were studied in intact human red cells loaded with Ca²⁺ by the aid of the ionophore A23187.After the complete elimination of the ionophore the passive Ca²⁺-permeability of the membrane returned to its normal low value, except when the intracellular Ca²⁺-concentration was higher than 3 mM or the ATP level fell below 100 μM. Within these limits the rate of Ca²⁺-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca²⁺]_i and reached a plateau at about 1 mM intracellular Ca²⁺-concentration. The maximum rate of Ca²⁺-efflux was about 85 μmol/l of cells per min at 37°C, pH 7.4. The activation energy of active Ca²⁺-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7.Ca²⁺-efflux was not connected with the counter-transport of cations.The Ca²⁺-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, $\text{N-}\text{ethylmaleimide}$ and $\text{p-}\text{chloromercuribenzoate}$ or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca²⁺-extrusion was brought about by 50–250 μM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca²⁺-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane.To examine the energy consumption of the Ca²⁺-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca²⁺-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na⁺-tetrathionate. Ca²⁺-activated ATPase activity connected with the Ca²⁺-pump was distinguished from other Ca²⁺-ATPase by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca²⁺-transported per ATP split was found to be $\text{2 : 1}$.     

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca²⁺-ATPase (PMCA), which is critical for maintaining a low intracellular Ca²⁺ concentration ([Ca²⁺]_i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca²⁺]_i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca²⁺]_i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca²⁺]_i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.     

Imaging of a glucose analog, calcium and NADH in neurons and astrocytes: dynamic responses to depolarization and sensitivity to pioglitazone

Neuronal Ca²⁺ dyshomeostasis associated with cognitive impairment and mediated by changes in several Ca²⁺ sources has been seen in animal models of both aging and diabetes. In the periphery, dysregulation of intracellular Ca²⁺ signals may contribute to the development of insulin resistance. In the brain, while it is well-established that type 2 diabetes mellitus is a risk factor for the development of dementia in the elderly, it is not clear whether Ca²⁺ dysregulation might also affect insulin sensitivity and glucose utilization. Here we present a combination of imaging techniques testing the disappearance of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as an indication of glycolytic activity in neurons and astrocytes. Our work shows that glucose utilization at rest is greater in neurons compared to astrocytes, and ceases upon activation in neurons with little change in astrocytes. Pretreatment of hippocampal cultures with pioglitazone, a drug used in the treatment of type 2 diabetes, significantly reduced glycolytic activity in neurons and enhanced it in astrocytes. This series of experiments, including Fura-2 and NADH imaging, provides results that are consistent with the idea that Ca²⁺ levels may rapidly alter glycolytic activity, and that downstream events beyond Ca²⁺ dysregulation with aging, may alter cellular metabolism in the brain.     

Regulation of rat brainstem tryptophan 5-monooxygenase. Calcium-dependent reversible activation by ATP and magnesium.

Tryptophan 5-monooxygenase in rat brainstem cytosol was activated about twofold by incubation with 0.5 mm ATP and 5 mm MgCl₂. The activation required micromolar concentrations of Ca²⁺ but was not dependent on either cyclic AMP or cyclic GMP. Rat brain cytosol was shown to possess an endogenous protein kinase which was markedly stimulated by the addition of Ca²⁺ using endogenous protein substrates. Following activation by ATP and Mg²⁺ in the presence of Ca²⁺, tryptophan 5-monooxygenase was reversibly deactivated to the original level by incubation at 30 °C after removal of Ca²⁺ by adding ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid and was then reactivated by incubation at 30 °C after subsequent addition of Ca²⁺ and ATP. The deactivation was markedly inhibited by the omission of Mg²⁺ or by the addition of NaF.     

Mitochondria Regulate Neutrophil Activation by Generating ATP for Autocrine Purinergic Signaling

Polymorphonuclear neutrophils (PMNs) form the first line of defense against invading microorganisms. We have shown previously that ATP release and autocrine purinergic signaling via P2Y₂ receptors are essential for PMN activation. Here we show that mitochondria provide the ATP that initiates PMN activation. Stimulation of formyl peptide receptors increases the mitochondrial membrane potential (Δψm) and triggers a rapid burst of ATP release from PMNs. This burst of ATP release can be blocked by inhibitors of mitochondrial ATP production and requires an initial formyl peptide receptor-induced Ca²⁺ signal that triggers mitochondrial activation. The burst of ATP release generated by the mitochondria fuels a first phase of purinergic signaling that boosts Ca²⁺ signaling, amplifies mitochondrial ATP production, and initiates functional PMN responses. Cells then switch to glycolytic ATP production, which fuels a second round of purinergic signaling that sustains Ca²⁺ signaling via P2X receptor-mediated Ca²⁺ influx and maintains functional PMN responses such as oxidative burst, degranulation, and phagocytosis.     

Glycolytic ATP Fuels the Plasma Membrane Calcium Pump Critical for Pancreatic Cancer Cell Survival

Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca²⁺ ([Ca²⁺]_i), as the ATP-dependent plasma membrane Ca²⁺ ATPase (PMCA) is critical for maintaining low [Ca²⁺]_i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca²⁺]_i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca²⁺]_i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.     

Lymphocyte calcium extrusion: kinetic and thermodynamic measurements using ratiometric dual-emission spectrofluorometry

A method is described for monitoring intracellular ionized calcium (Ca²⁺) and determining kinetic and thermodynamic parameters of Ca⁺-extrusion from intact lymphocytes. The method uses ratiometric spectrofluorometry and the fluorescent Ca²⁺ dye indo-1. Lymphocytes were loaded with calcium and placed in a low calcium medium. A novel formula for calculation of intracellular Ca^2– that corrects for background fluorescence and fluorescence quenching was used. Calcium extrusion resulted in exponential decrease in cytoplasmic Ca²⁺ with a rate constant of 0.031 ± 0.003 sec^–1, maximal rate of 23 ± 7 nM/sec, dissociation constant of 366 ± 63 nM, Hill coefficient of 2.3 ± 0.4, Q₁₀ of 2.58 ± 0.28, and activation energy of 18.3 Kcal/mol. This method should allow for characterization of the Ca²⁺-extrusion system of lymphocytes and may be applicable to other blood cell types.Abbreviations DMSO dimethyl sulfoxide - HEPES [N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid],sodium salt - Indo-1/AM acetoxymethyl ester of indo-1 - IP₃ inositol 1,4,5-triphosphate - IP₄ inositol 1,3,4,5-tetrakisphosphate - RPMI Roswell Park Memorial Institute — 1640 culture medium - TPEN tetrakis-[2-pyridylmethyl]-ethylenediamine     

Distinctive Features of Catalytic and Transport Mechanisms in Mammalian Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases

Ca²⁺ (sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA)) and Cu⁺ (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca²⁺, demonstrated by the addition of ATP and Ca²⁺ to SERCA deprived of Ca²⁺ (E2) as compared with ATP to Ca²⁺-activated enzyme (E1·2Ca²⁺). Activation by Ca²⁺ is slower at low pH (2H⁺·E2 to E1·2Ca²⁺) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca²⁺ translocation. A “H⁺-gated pathway,” demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca²⁺ release by H⁺/Ca²⁺ exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu⁺/H⁺ exchange. As opposed to SERCA after Ca²⁺ chelation, ATP7A/B does not undergo reverse phosphorylation with P_i after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.     

The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin,ATP and partial proteolysis

(1) Depending on the assay conditions, the ability of the Ca²⁺-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg²⁺ and K⁺ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. (2) In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca²⁺ so that the activity becomes negligible above 30 μM Ca²⁺. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca²⁺. (3) In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca²⁺ are needed and phosphatase activity is maximal in media without Ca²⁺. This is the first report of an activity sustained by the Ca²⁺-ATPase of red cell membranes in the absence of Ca²⁺. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca²⁺. These results strongly suggest that during activation by calmodulin, Ca²⁺ is needed only to form the calmodulin-Ca²⁺ complex which is the effective cofactor. (4) Protection by ATP of the inhibitory effects of Ca²⁺ and the induction of phosphatase activity by ATP + Ca²⁺ suggests that activation of the phosphatase by Ca²⁺ in media with ATP requires the combination of the cation at sites in the ATPase. (5) Results can be rationalized assuming that E₂, the conformer of the Ca²⁺-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca²⁺ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E₁ and E₂ towards E₂. On the other hand, ATP + Ca²⁺ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E₂ predominates over other conformers of the ATPase.     

Mitochondrial Generation of Oxygen Radicals During Reoxygenation of Ischemic Tissues

Ischemia and reperfusion causes severe mitochondrial damage, including swelling and deposits of hyd-roxyapatite crystals in the mitochondrial matrix. These crystals are indicative of a massive influx of Ca²⁺ into the mitochondrial matrix occurring during reoxygenation. We have observed that mitochondria isolated from rat hearts after 90 minutes of anoxia followed by reoxygenation, show a specific inhibition in the electron transport chain between NADH dehydrogenase and ubiquinone in addition to becoming uncoupled (unable to generate ATP). This inhibition is associated with an increased H₂O₂ formation at the NADH dehydrogenase level in the presence of NADH dependent substrates. Control rat mitochondria exposed for 15 minutes to high Ca²⁺ (200 nmol/mg protein) also become uncoupled and electron transport inhibited between NADH dehydrogenase and ubiquinone. a lesion similar to that observed in post-ischem-ic mitochondria. This Ca²⁺ -dependent effect is time dependent and may be partially prevented by albumin, suggesting that it may be due to phospholipase A₂ activation. releasing fatty acids, leading to both inhibition of electron transport and uncoupling. Addition of arachidonic or linoleic acids to control rat heart mitochondria, inhibits electron transport between Complex I and III. These results are consistent with the following hypothesis: during ischemia, the intracellular energy content drops severely, affecting the cytoplasic concentration of ions such as Na⁺ and Ca²⁺. Upon reoxygenation, the mitochondrion is the only organelle capable of eliminating the excess cytoplasmic Ca²⁺ through an electrogenic process requiring oxygen (the low ATP concentration makes other ATP-dependent Ca^?' lransport systems non-operational). Ca²⁺-overload of mitochondria activates phospholipase A₂ releasing free fatty acids, leading to uncoupling and inhibition of the interactions between Complex I and III of the respiratory chain. As a consequence, the NADH-dehydrogenase becomes highly reduced, and transfers electrons directly to oxygen generating O₂.     

Separation of the Mg-ATPases from the Ca-Phosphatase Activity of Microsomal Membranes Prepared from Barley Roots

Two methods for preparing membrane fractions from barley (Hordeum vulgare cv California Mariout 72) roots were compared in order to resolve reported differences between the characteristics of the plasma membrane ATPase of barley and that of other species. When microsomal membranes were prepared by a published procedure and applied to a continuous sucrose gradient, the membranes sedimented as a single broad band with a peak density of 1.16 grams per cubic centimeter (g/cm³). Activities of NADH cytochrome (Cyt) c reductase, Ca²⁺-ATPase, and Mg²⁺-ATPase were coincident and there was little ATP-dependent proton transport anywhere on the gradient. When the homogenization procedure was modified by increasing the pH of the buffer and the ratio of buffer to roots, the microsomal membranes separated as several components on a continuous sucrose gradient. A Ca²⁺-phosphatase was at the top of the gradient, NADH Cyt c reductase at 1.08 g/cm³, a peak of ATP-dependent proton transport at 1.09 to 1.12 g/cm³, a peak of nitrate-inhibited ATPase at 1.09 to 1.12 g/cm³, and of vanadate-inhibited ATPase at 1.16 g/cm³. The Ca²⁺-phosphatase had no preference for ATP over other nucleoside di- and tri-phosphates and was separated from the vanadate-inhibited ATPase on a sucrose gradient; approximately 70% of the Ca²⁺-phosphatase was removed from the microsomes by washing with 150 millimolar KCl. The vanadate-sensitive ATPase required Mg²⁺, was highly specific for ATP, and was not affected by the KCl wash. These results show that barley roots have a plasma membrane ATPase similar to that of other plant species.     

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca²⁺-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca²⁺ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca²⁺-containing but not in a Ca²⁺-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y₂ receptors and PI3K/Akt pathway activation involving Ca²⁺, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca²⁺ influx/CaM pathway.     

Mitochondrial calcium as a key regulator of mitochondrial ATP production in mammalian cells

Mitochondrial Ca²⁺ transport was initially considered important only in buffering of cytosolic Ca²⁺ by acting as a “sink” under conditions of Ca²⁺ overload. The main regulator of ATP production was considered to be the relative concentrations of high energy phosphates. However, work by Denton and McCormack in the 1970s and 1980s showed that free intramitochondrial Ca²⁺ ([Ca²⁺]_m) activated dehydrogenase enzymes in mitochondria, leading to increased NADH and hence ATP production. This leads them to propose a scheme, subsequently termed a “parallel activation model” whereby increases in energy demand, such as hormonal stimulation or increased workload in muscle, produced an increase in cytosolic [Ca²⁺] that was relayed by the mitochondrial Ca²⁺ transporters into the matrix to give an increase in [Ca²⁺]_m. This then stimulated energy production to meet the increased energy demand. With the development of methods for measuring [Ca²⁺]_m in living cells that proved [Ca²⁺]_m changed over a dynamic physiological range rather than simply soaking up excess cytosolic [Ca²⁺], this model has now gained widespread acceptance. However, work by ourselves and others using targeted probes to measure changes in both [Ca²⁺] and [ATP] in different cell compartments has revealed variations in the interrelationships between these two in different tissues, suggesting that metabolic regulation by Ca²⁺ is finely tuned to the demands and function of the individual organ.     

Purinergic signalling is involved in the malaria parasite <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca²⁺ levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca²⁺ increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca²⁺ levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca²⁺ in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca²⁺]_c. Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca²⁺]_c in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.     

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na⁺/Ca²⁺ exchanger (NCX) is the major Ca²⁺ efflux pathway on the sarcolemma, counterbalancing Ca²⁺ influx via L-type Ca²⁺ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca²⁺ load and can contribute to abnormal Ca²⁺ handling and arrhythmias. NADH/NAD⁺ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD⁺ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (I_NCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced I_NCX inhibition was abolished by the H₂O₂ scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because I_NCX was insensitive to cytosolic NADPH, and NADH-induced ROS and I_NCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD⁺ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca²⁺ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.     

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1)

Ryanodine receptor type 1 (RyR1) releases Ca²⁺ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca²⁺, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca²⁺, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [³H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca²⁺-binding site to sensitize RyR1 to Ca²⁺. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca²⁺. However, enhanced RyR1 activation occurred in the presence of Ca²⁺, AMPPCP, and Caf. In the absence of Ca²⁺, Na⁺ inhibited [³H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca²⁺-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca²⁺, Na⁺ is predicted to inhibit RyR1 by interacting with the Ca²⁺-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca²⁺-binding site, and conversely, Ca²⁺ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca²⁺, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca²⁺-, ATP-, and Caf-binding sites.     

Modulation of the kinetic characteristics of the sarcoplasmic reticulum ATPase by membrane fluidity

(Ca²⁺ + Mg²⁺)-ATPase from sarcoplasmic reticulum has been reconstituted with dipalmitoylphosphatidylcholine, and the activating effect of ATP and Ca²⁺ on this enzyme has been studied at different temperatures. It has been found that two kinetic forms of the enzyme are interconverted at about 31°C, and this is possibly related to a phase change in the phospholipid which is more directly associated with the protein. Above 31°C the enzyme is less dependent on ATP activation at high ATP concentrations but shows positive cooperativity for Ca²⁺ activation. On the other hand, below 31°C, the reconstituted enzyme is more dependent on ATP for activation at high ATP concentrations than the purified ATPase and does not show cooperativity for Ca²⁺ activation.