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.     

Epigallocatechin-3-gallate elicits Ca spike in MCF-7 breast cancer cells: Essential role of Cav3.2 channels

We used MCF-7 human breast cancer cells that endogenously express Cav3.1 and Cav3.2 T-type Ca²⁺ channels toward a mechanistic study on the effect of EGCG on [Ca²⁺]_i. Confocal Ca²⁺ imaging showed that EGCG induces a [Ca²⁺]_i spike which is due to extracellular Ca²⁺ entry and is sensitive to catalase and to low-specificity (mibefradil) and high-specificity (Z944) T-type Ca²⁺channel blockers. siRNA knockdown of T-type Ca²⁺ channels indicated the involvement of Cav3.2 but not Cav3.1. Application of EGCG to HEK cells expressing either Cav3.2 or Cav3.1 induced enhancement of Cav3.2 and inhibition of Cav3.1 channel activity. Measurements of K⁺ currents in MCF-7 cells showed a reversible, catalase-sensitive inhibitory effect of EGCG, while siRNA for the Kv1.1 K⁺ channel induced a reduction of the EGCG [Ca²⁺]_i spike. siRNA for Cav3.2 reduced EGCG cytotoxicity to MCF-7 cells, as measured by calcein viability assay. Together, data suggest that EGCG promotes the activation of Cav3.2 channels through K⁺ current inhibition leading to membrane depolarization, and in addition increases Cav3.2 currents. Cav3.2 channels are in part responsible for EGCG inhibition of MCF-7 viability, suggesting that deregulation of [Ca²⁺]_i by EGCG may be relevant in breast cancer treatment.     

Ca2+ influx and clearance at hyperpolarized membrane potentials modulate spontaneous and stimulated exocytosis in neuroendocrine cells

Neuroendocrine adrenal chromaffin cells release neurohormones catecholamines in response to Ca²⁺ entry via voltage-gated Ca²⁺ channels (VGCCs). Adrenal chromaffin cells also express non-voltage-gated channels, which may conduct Ca²⁺ at negative membrane potentials, whose role in regulation of exocytosis is poorly understood. We explored how modulation of Ca²⁺ influx at negative membrane potentials affects basal cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in metabolically intact voltage-clamped bovine adrenal chromaffin cells. We found that in these cells, Ca²⁺ entry at negative membrane potentials is balanced by Ca²⁺ extrusion by the Na⁺/Ca²⁺ exchanger and that this balance can be altered by membrane hyperpolarization or stimulation with an inflammatory hormone bradykinin. Membrane hyperpolarization or application of bradykinin augmented Ca²⁺-carrying current at negative membrane potentials, elevated basal [Ca²⁺]_i, and facilitated synchronous exocytosis evoked by the small amounts of Ca²⁺ injected into the cell via VGCCs (up to 20 pC). Exocytotic responses evoked by the injections of the larger amounts of Ca²⁺ via VGCCs (> 20 pC) were suppressed by preceding hyperpolarization. In the absence of Ca²⁺ entry via VGCCs and Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger, membrane hyperpolarization induced a significant elevation in [Ca²⁺]_i and asynchronous exocytosis. Our results indicate that physiological interferences, such as membrane hyperpolarization and/or activation of non-voltage-gated Ca²⁺ channels, modulate basal [Ca²⁺]_i and, consequently, segregation of exocytotic vesicles and their readiness to be released spontaneously and in response to Ca²⁺ entry via VGCCs. These mechanisms may play role in homeostatic plasticity of neuronal and endocrine cells.     

Metabolic regulation of the PMCA: Role in cell death and survival

The plasma membrane Ca²⁺-ATPase (PMCA) is a ubiquitously expressed, ATP-driven Ca²⁺ pump that is critical for maintaining low resting cytosolic Ca²⁺ ([Ca²⁺]_i) in all eukaryotic cells. Since cytotoxic Ca²⁺ overload has such a central role in cell death, the PMCA represents an essential “linchpin” for the delicate balance between cell survival and cell death. In general, impaired PMCA activity and reduced PMCA expression leads to cytotoxic Ca²⁺ overload and Ca²⁺ dependent cell death, both apoptosis and necrosis, whereas maintenance of PMCA activity or PMCA overexpression is generally accepted as being cytoprotective. However, the PMCA has a paradoxical role in cell death depending on the cell type and cellular context. The PMCA can be differentially regulated by Ca²⁺-dependent proteolysis, can be maintained by a localised glycolytic ATP supply, even in the face of global ATP depletion, and can be profoundly affected by the specific phospholipid environment that it sits within the membrane. The major focus of this review is to highlight some of the controversies surrounding the paradoxical role of the PMCA in cell death and survival, challenging the conventional view of ATP-dependent regulation of the PMCA and how this might influence cell fate.     

Differential Regulation of Calcium-Activated Potassium Channels by Dynamic Intracellular Calcium Signals

Calcium (Ca²⁺)-activated K⁺ (K_Ca) channels regulate membrane excitability and are activated by an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), leading to membrane hyperpolarization. Most patch clamp experiments that measure K_Ca currents use steady-state [Ca²⁺] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca²⁺]_i changes dynamically, for example during [Ca²⁺]_i oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca²⁺]_i on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each K_Ca subtype in isolation were used to simultaneously measure agonist-evoked [Ca²⁺]_i signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca²⁺]_i oscillations induced a corresponding K_Ca current that faithfully followed the [Ca²⁺]_i in 13–50% of cells, suggesting a good synchronization. However, [Ca²⁺]_i and K_Ca current was much less synchronized in 50–76% of cells that exhibited Ca²⁺-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca²⁺]_i events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca²⁺]_i and K_Ca current was least synchronized, 36% of total [Ca²⁺]_i spikes occurred without activating a corresponding K_Ca current spike, suggesting that BK_Ca channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca²⁺]_i and K_Ca current suggests that this relationship is more complex than could be predicted from steady-state [Ca²⁺]_i and K_Ca current. These phenomena may be important for encoding stimulus–response coupling in various cell types.     

Capacitation suppression by mouse seminal vesicle autoantigen involves a decrease in plasma membrane Ca2+‐ATPase (PMCA)‐mediated intracellular calcium

Successful fertilization is tightly regulated by capacitation and decapacitation processes. Without appropriate decapacitation regulation, sperm would undergo a spontaneous acrosome reaction which leads to loss of fertilization ability. Seminal plasma is known to negatively regulate sperm capacitation. However, the suppressive mechanisms still remain unclear. In this study, we demonstrate the decapacitation mechanism of mouse seminal vesicle autoantigen (SVA) might target membrane sphingomyelin (SPM) and regulate plasma membrane Ca²⁺‐ATPase (PMCA) activity. The SVA was shown to suppress sperm capacitation induced by a broad panel of capacitation factors (bovine serum albumin (BSA), PAF, and cyclodextrin (CD)). Furthermore, SVA significantly decreased [Ca²⁺]_i and NaHCO₃‐induced [cAMP]_i. Cyclic AMP agonists bypassed the SVA's suppressive ability. Importantly, the SVA may regulate PMCA activity which was evidenced by the fact that the SVA decreased the [Ca²⁺]_i and intracellular pH (pH_i) of sperm; meanwhile, a PMCA inhibitor (carboxyeosin) could reverse SVA's suppression of [Ca²⁺]_i. The potential target of the SVA on membrane SPM/lipid rafts was highlighted by the high binding affinity of SPM–SVA (with a K_d of ～3 µM) which was close to the IC₅₀ of SVA's suppressive activity. Additionally, treatment of mink lung epithelial cells with the SVA enhanced plasminogen activator inhibitor (PAI)‐1 expression stimulated by tumor growth factor (TGF)‐β and CD. These observations supported the membrane lipid‐raft targeting of SVA. In summary, in this paper, we demonstrate that the decapacitation mechanism of the SVA might target membrane sphingolipid SPM and regulate PMCA activity to lower [Ca²⁺]_i, thereby decreasing the [cAMP]_i level and preventing sperm pre‐capacitation. J. Cell. Biochem. 111: 1188–1198, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of Phenylalanine and its Metabolites on Cytoplasmic Free Calcium in Cortical Neurons

Classic phenylketonuria (PKU) is characterized by brain lesions. However, its underlying neurotoxic mechanisms remain unknown. Based on our previous studies, we hypothesized that calcium might participate in PKU-associated neuropathy. In cultured cortical neurons, cytoplasmic free calcium concentration ([Ca²⁺]_i) decreased dramatically when treatment with phenylalanine (Phe) and phenyllactic acid, while phenylacetic acid treatment immediately increased [Ca²⁺]_i, which began to decrease after 3 min. Moreover, [Ca²⁺]_i decreased dramatically after Phe treatment in the presence of EGTA suggesting that Phe might increase [Ca²⁺]_i efflux. Phe-induced [Ca²⁺]_i decrease was strongly inhibited by vanadate, a non-specific plasma membrane Ca²⁺-ATPase (PMCA) antagonist, suggesting that Phe might increase [Ca²⁺]_i efflux throught modulating PMCA. These findings were further supported by the facts that Phe could increase membrance ⁴⁵Ca-uptake capability and PMCA activity. In contrast, treatment of KBR7943 or thapsigargin, antagonists to Na/Ca Exchanger (NCX) and Sarco/Endoplasmic reticulum Ca²⁺-ATPase (SERCA), respectively, did not elicit any changes in [Ca²⁺]_i. Specific siRNA against PMCA had an effect similar to vanadate. Since the brain injury induced by phenylalaninemia was thought to be a chronic process, we cultured cortical neurons in the presence of Phe for 2 weeks and measured [Ca²⁺]_i, PMCA activity and ⁴⁵Ca-uptake capability at days 3, 7, 9 and 14, respectively. PMCA activity and ⁴⁵Ca-uptake capability decreased from day 9, at the same time [Ca²⁺]_i increase was observed. In conclusion, PMCA participate in regulating Phe-induced initial rapid decrease in [Ca²⁺]_i and subsequent long-term increase in [Ca²⁺]_i.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.     

Characteristics of cytosolic Ca2+ elevation induced by muscarinic receptor activation in single adrenal chromaffin cells of the guinea pig

In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca²⁺]_i) followed by a sustained rise. In Ca²⁺-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca²⁺]_i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of −60 mV, the muscarine-induced [Ca²⁺]_i, rise, especially its sustained phase, decreased in magnitude. intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca²⁺]_i and inhibited the following [Ca²⁺]_i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca²⁺]_i. Muscarine-induced sustained [Ca²⁺]_i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca²⁺]_i rise and Mn²⁺ influx in response to muscarine were suppressed by a voltage-dependent Ca²⁺ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca²⁺ entry, but also Ca²⁺ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca²⁺ channels may function as one of the Ca²⁺ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells.     

Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction

Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pH_i) and Ca²⁺ ([Ca²⁺]_i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K⁺ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K⁺ or by addition of solubilized zona pellucida (sZP). Moreover, K⁺ and sZP were also able to increase [Ca²⁺]_i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K⁺ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca²⁺]_i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction.     

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands

Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺]_i in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺]_i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺]_i increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺]_i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺]_i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺]_i, although UTP (a P2Y_2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺]_i, but α,β-meATP (a P2X_1,3 receptor agonist) had no effect. RT-PCR indicated that P2X_2,3,4,5,6,7 and P2Y_1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.     

Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

Plasma membrane Ca²⁺-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca²⁺]_i). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca²⁺]_i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.     

Plasma Membrane Ca2+-ATPase Isoforms Composition Regulates Cellular pH Homeostasis in Differentiating PC12 Cells in a Manner Dependent on Cytosolic Ca2+ Elevations

Plasma membrane Ca²⁺-ATPase (PMCA) by extruding Ca²⁺ outside the cell, actively participates in the regulation of intracellular Ca²⁺ concentration. Acting as Ca²⁺/H⁺ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca²⁺ overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pH_mito and pH_cyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca²⁺ clearance and partially attenuated cellular acidification during KCl-stimulated Ca²⁺ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca²⁺ overload. Cyclosporin and bongkrekic acid prevented Ψ_m loss suggesting the involvement of Ca²⁺-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.     

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.     

Mechanotransduction in Colonic Smooth Muscle Cells

We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca²⁺] _i ) with peak of 422.7 ± 43.8 nm above an average resting [Ca²⁺] _i of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca²⁺] _i increase was reduced by 50% in the absence of extracellular Ca²⁺, while the prolonged [Ca²⁺] _i recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride (100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca²⁺] _i recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca²⁺] _i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca²⁺] _i transient peak. [Ca²⁺] _i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca²⁺-free solution, or when Ca²⁺ stores were depleted by thapsigargin in Ca²⁺-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca²⁺] _i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca²⁺ store. Received: 24 March 1997/Revised: 21 July 1997     

Taurine prevents intracellular calcium overload during calcium paradox of cultured cardiomyocytes

Summary The effect of taurine on the cellular distribution of [Ca²⁺]_i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca²⁺ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca²⁺-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca²⁺]; rapidly fell to a level of 3–6 × 10^–8M. Subsequent restoration of medium calcium increased [Ca²⁺]_i to level 4–7 times normal. Large increases in [Ca²⁺]_i were observed in most cells and were associated with the development of contracture and bleb formation.Taurine pretreatment (20mM) caused no significant effect on [Ca²⁺]_i during Ca²⁺ depletion. However, it inhibited excessive accumulation of [Ca²⁺]_i during the Ca²⁺ repletion. Moreover, taurine treated cells recovered their Ca²⁺-transients and beating pattern earlier than non-treated cells. Finally morphological abnormalities commonly associated with calcium overload were attenuated by taurine treatment.     

Insulin Protects Pancreatic Acinar Cells from Palmitoleic Acid-induced Cellular Injury

Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca²⁺ ([Ca²⁺]_i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca²⁺]_i are linked critically by the ATP-driven plasma membrane Ca²⁺-ATPase (PMCA) important for maintaining low resting [Ca²⁺]_i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca²⁺]_i was measured by fura-2 imaging. An in situ [Ca²⁺]_i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca²⁺ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca²⁺ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca²⁺ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca²⁺ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.     

Delayed activation of plasma membrane Ca pump in human neutrophils

The interplay between Ca²⁺ efflux mechanisms of the plasma membrane (PM) and transient changes of the cytosolic concentration of ionized calcium ([Ca²⁺]_i) was studied in suspensions of human neutrophils loaded with the [Ca²⁺]_i indicator, Fura-2. To reveal Ca²⁺ efflux through PM the interference of intracellular Ca stores was prevented by preincubating the cells in the presence of EGTA, thapsigargin, and ionomycin. Addition of econazole prevented varying entry of divalent cations regulated by the filling state of Ca stores. The preincubation seemed to empty and permeabilize virtually all Ca stores, ensuring that the monitored changes of [Ca²⁺]_i were caused exclusively by PM Ca²⁺ transporters. Following preincubation, the addition of CaCl₂ induced, mediated by ionomycin, a transient rise of [Ca²⁺]_i, a spike, eventually decreasing to an intermediary [Ca²⁺]_i level. The ATP-dependent decrease of [Ca²⁺]_i terminating the spike was abolished by the calmodulin antagonist, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7), but not by the protein kinase C inhibitor, staurosporine, nor by Na⁺-free medium, suggesting that neither activity of protein kinase C nor exchange was necessary for generation of the Ca²⁺ spike. In conclusion, the PM Ca²⁺ pump was responsible for the Ca²⁺ spike by responding to the rapid rise of [Ca²⁺]_i by a delayed activation, possibly involving calmodulin. This characteristic feature of the PM pump may be important for the generation of cellular [Ca²⁺]_i spikes in general.     

Purinergic signalling in rat GFSHR-17 granulosa cells: an <Emphasis Type="Italic">in vitro</Emphasis> model of granulosa cells in maturing follicles

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca²⁺-imaging and perforated patch-clamp. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of −40 mV. This was consistent with high K⁺− and Cl⁻ permeability and a high intracellular Cl⁻ concentration of 40 mM. Application of ATP for 5–15 s every 3 min induced repeated [Ca²⁺]_i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP₃-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y₂ and P2Y₄ receptors and that the [Ca²⁺]_i increase was a prerequisite for hyperpolarization. Inhibitors of Ca²⁺-activated channels or K⁺ channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl⁻ channels hyperpolarized cells to −70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y₂ and P2Y₄ receptors in granulosa cells modulate Cl⁻ permeability by regulating Ca²⁺-release.