The calmodulin-activated form of the Ca2+-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%

The thermodynamic efficiency of the calmodulin-activated form of the Ca²⁺-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca²⁺ concentration ([Ca²⁺]_i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][P_i]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg²⁺, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca²⁺]_o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM P_i, variable [ADP]. Under these conditions, with the pump working near itsK _m of 64nM, the [Ca²⁺]_i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca²⁺]_i/[Ca²⁺]_o ratio against [ATP]/([ADP][P_i]) gave a straight line with a slope of 1.5×10⁷M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×10⁷M). These results demonstrate (1) tight coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2 Ca²⁺ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC₅₀ of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.     

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent

The thermodynamic efficiency of the Ca²⁺-Mg²⁺-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca²⁺ gradient established with the ATP/(ADP*P_i) ratio. The evaluation was made at an external Ca²⁺ level (4.7 × 10^–8 M) which is below theK _m value of 7 × 10^–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca²⁺ concentration of 17 mM was observed at infinite ATP/(ADP*P_i) ratio (absence of ADP). This corresponds to a [Ca²⁺]_i/[Ca²⁺]₀ gradient of 3.6 × 10⁵. A Ca²⁺ gradient one-half as large was observed at an ATP/(ADP*P_i) ratio of 3.5 × 10³ M^–1. The square of the Ca²⁺ gradient is shown to be proportional to the ATP/(ADP*P_i) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 10⁶ M) under these conditions (0.1 mM Mg²⁺, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.     

ATP-Induced Oscillations of Cytosolic Ca2+ Activity in Cultured Astrocytes from Rat Brain are Modulated by Medium Osmolarity Indicating a Control of [Ca2+]i Oscillations by Cell Volume

Oscillations of cytosolic Ca²⁺ activity ([Ca²⁺]_i) induced by stimulation with ATP in rat astrocytes in primary cultures were analysed. Astrocytes, prepared from the brains of newborn rats, loaded with the fluorescent Ca²⁺ indicator fura-2/AM, were continuously stimulated with ATP (10 M). ATP caused a large initial [Ca²⁺ peak, followed by regular [Ca²⁺]_i oscillations (frequencies 1–5/min). Astrocytes were identified by glial fibrillary acidic protein staining of cells after [Ca²⁺]_i recording. The oscillations were reversibly blocked by the P₂ purinoceptor antagonist suramin (30 M). Influx of extracellular Ca²⁺ and mobilization of Ca²⁺ from intracellular stores both contributed to the oscillations. The effects of hypertonic and hypotonic superfusion medium on ATP-induced [Ca²⁺]_i oscillations were examined. Hypertonic medium (430 mOsm) reversibly suppressed the ATP-induced oscillations. Hypotonic medium (250 mOsm), in spite of having heterogeneous effects, most frequently induced a rise in [Ca²⁺]_i, or reversibly increased the frequency of the oscillations. Thus, a change in cell volume might be closely connected with [Ca²⁺]_i oscillations in astrocytes indicating that [Ca²⁺]_i oscillations in glial cells play an important role in regulatory volume regulation in the brain.     

Spatial and temporal patterns of intracellular calcium in colonic smooth muscle

Summary Intracellular calcium [Ca²⁺] _i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca²⁺] _i transient followed by a steady-state increase as the characteristic [Ca²⁺] _i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca²⁺] _i signal in single cells. The distribution of [Ca²⁺] _i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca²⁺] _i present in the subplasmalemmal space and in one cell pole. [Ca²⁺] _i gradients within these regions were not constant but showed temporal changes in the form of [Ca²⁺] _i oscillations and spatial changes in the form of [Ca²⁺] _i waves. [Ca²⁺] _i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca²⁺] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca²⁺], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca²⁺] _i waves was also independent of influx of extracellular Ca²⁺. [Ca²⁺] _i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca²⁺] _i induced by different agonists were encoded into changes of baseline [Ca²⁺] _i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca²⁺] _i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca²⁺] _i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca²⁺] _i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar⁹,Met(O₂)¹¹]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.     

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) in C₆ glioma cells with an EC₅₀ of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca²⁺]_i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca²⁺ and Na⁺ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca²⁺ channels (Co²⁺, Mn²⁺, La³⁺, or Cd²⁺). In Ca²⁺-free medium, ATP caused only a transient increase in [Ca²⁺]_i as opposed to a sustained [Ca²⁺]_i increase in normal medium. The ATP-induced elevation of [Ca²⁺]_i was resistant to Na⁺ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La³⁺. The differences in the characteristics of ATP-caused P₁ hydrolysis and [Ca²⁺]_i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca²⁺ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P₁-purinergic receptors, the C₆ glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na⁺]_o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - IP₄ inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

Negative Regulation of CFTR Activity by Extracellular ATP Involves P2Y2 Receptors in CFTR-expressing CHO Cells

Extracellular nucleotides exert autocrine/paracrine effects on ion transport by activating P2 receptors. We studied the effects of extracellular ATP and UTP on the cystic fibrosis transmembrane conductance regulator (CFTR) channel stably expressed in Chinese Hamster Ovary cells (CHO-BQ1 cells). CFTR activity was measured using the (¹²⁵I) iodide efflux technique and whole-cell patch-clamp recording in response to either forskolin or xanthine derivatives. Using RT-PCR and intracellular calcium concentration ([Ca²⁺]_i) measurement, we showed that CHO-BQ1 cells express P2Y2 but not P2Y4 receptors. While ATP and UTP induced similar increases in [Ca²⁺]_i, pre-addition by one of these two agonists desensitized the response for the other, suggesting that ATP- and UTP-induced [Ca²⁺]_i increases were mediated by a common receptor, which was identified as the P2Y2 subtype. CFTR activity was reduced by ATP and UTP but not by ADP or adenosine applications. This inhibitory effect of ATP on CFTR activity was not due to a change in cAMP level. Furthermore, CFTR activation by forskolin or IBMX failed to promote [Ca²⁺]_i increase, suggesting that CFTR activation did not generate an ATP release large enough to stimulate P2Y2 receptors. Taken together, our results show that endogenous P2Y2 receptor activation downregulates CFTR activity in a cAMP-independent manner in CHO cells. B. Marcet and V. Chappe contributed equally to this work.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5′-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5′-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca²⁺]_i) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca²⁺]_i transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca²⁺]_i transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca²⁺]_i response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca²⁺]_i transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9468-1) contains supplementary material, which is available to authorized users.     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

The mechanism of protriptyline-induced Ca2+ movement and non-Ca2+-triggered cell death in PC3 human prostate cancer cells

Abstract

Protriptyline, a tricyclic anti-depressant, is used primarily to treat the combination of symptoms of anxiety and depression. However, the effect of protriptyline on prostate caner is unknown. This study examined whether the anti-depressant protriptyline altered Ca²⁺ movement and cell viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i. Protriptyline evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Protriptyline-evoked Ca²⁺ entry was inhibited by store-operated channel inhibitors (nifedipine, econazole and SKF96365), protein kinase C activator (phorbol 12-myristate 13 acetate, PMA) and protein kinase C inhibitor (GF109203X). Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydr-oquinone (BHQ) in Ca²⁺-free medium inhibited 60% of protriptyline-evoked [Ca²⁺]_i rises. Conversely, treatment with protriptyline abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 suppressed 50% of protriptyline-evoked [Ca²⁺]_i rises. At concentrations of 50–70?µM, protriptyline decreased cell viability in a concentration-dependent manner; which were not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, in PC3 cells, protriptyline evoked [Ca²⁺]_i rises by inducing phospholipase C-associated Ca²⁺ release from the endoplasmic reticulum and other stores, and Ca²⁺ influx via protein kinase C-sensitive store-operated Ca²⁺ channels. Protriptyline caused cell death that was independent of [Ca²⁺]_i rises.     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Neuronal Control of Exocytosis and Calcium Homeostasis

We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca²⁺ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca²⁺-free external solution caused a [Ca²⁺] _i increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca²⁺ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated.     

The Nootropic Drug Vinpocetine Inhibits Veratridine-Induced [Ca2+]i Increase in Rat Hippocampal CA1 Pyramidal Cells

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3,16-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca²⁺]_i, this study investigated the effect of vinpocetine on [Ca²⁺]_i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na⁺ influx, causing depolarization and increasing [Ca²⁺]_i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca²⁺]_i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 M) significantly inhibited the elevation in [Ca²⁺]_i induced by veratridine (10 M). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca²⁺]_i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca²⁺]_i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.     

Effects of caffeine and raynodine on low pHi-induced changes in gap junction conductance and calcium concentration in crayfish septate axons

Summary Electrical uncoupling of crayfish septate axons with acidification has been shown to cause a substantial increase in [Ca²⁺]_i which closely matches in percent the increase in junctional resistance. To determine the origin of [Ca²⁺]_i increase, septate axons have been exposed either to drugs that influence Ca²⁺ release from internal stores, caffeine and ryanodine, or to treatments that affect Ca²⁺ entry. A large increase in junctional resistance and [Ca²⁺]_i maxima above controls resulted from addition of caffeine (10–30mm) to acetate solutions, while a substantial decrease in both parameters was observed when exposure to acetate-caffeine was preceded by caffeine pretreatment. In contrast, ryanodine (1–10 m) always caused a significant decrease in junctional resistance and [Ca²⁺]_i maxima when applied either together with acetate or both before and with acetate. Calcium channel blockers such as La³⁺, Cd²⁺ and nisoldipine had no effect, while an increase in the [Ca²⁺] of acetate solutions either decreased junctional resistance and [Ca²⁺]_i maxima or had no effect. The data suggest that cytoplasmic acidification causes an increase in [Ca²⁺]_i by releasing Ca²⁺ from caffeine and ryanodine-sensitive Ca²⁺ stores. The increase in [Ca²⁺]_i results in a decrease in gap junction conductance.     

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet

Summary This communication reports the kinetics of the Na⁺/ Ca²⁺ exchanger and of the plasma membrane (PM) Ca²⁺ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca²⁺ extrusion and its Na⁺ dependence for concentrations of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in the 1–10-m range. The PM Ca²⁺ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca²⁺]_cyt] 1.5 m with the fluorescent Ca²⁺ indicator quin2 (K _d= 115 nm). That study determined that the PM Ca²⁺ pump in the basal state has a V _max = 0.098 mm/min, a K _m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca²⁺]_cyt with the intracellular Ca²⁺ probe, rhod2 (K _d= 500 nm), which has almost a fivefold lower affinity for Ca²⁺. An Appendix also describes the Mg²⁺ and pH dependence of the K _dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca²⁺ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca²⁺ extrusion into a Ca²⁺-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca²⁺ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca²⁺ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca²⁺ with a K _m= 0.97 ± 0.31 m and V_max = 1.0 ± 0.6 mm/ min. (ii) At high [Ca²⁺]_cyt, the exchanger works at a rate 10 times as large as the basal V _max of the PM Ca²⁺ extrusion pump. (iii) The exchanger can work in reverse after Na⁺ loading of the cytoplasm by monensin. (iv) The PM Ca²⁺ extrusion pump is activated by exposure to [Ca²⁺]_cyt 1.5 m for 20–50 sec. Activation raises the pump V _max to 1.6 ± 0.6 mm/min and the K _mto 0.55 ± 0.24 m. (v) The Ca²⁺ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca²⁺]_cyt. In summary, the results show that the human platelet can extrude Ca²⁺ very rapidly at high [Ca²⁺]_cyt. Both the Na⁺/Ca²⁺ exchanger and Ca²⁺ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca²⁺ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca²⁺]_cyt cytoplasmic Ca²⁺ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or V_extrusion true rate of Ca²⁺ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - _I fraction of high-affinity rhod2 complexed with Ca²⁺ - F the observed fluorescence - F_min the minimal fluorescence observed in the absence of Ca²⁺ - F_max the maximal fluorescence observed when the dye is saturated with Ca²⁺ - X₁ the fraction of high-affinity dye - K _d,1 dissociation constant of high-affinity dye - K _d,2 dissociation constant of the low-affinity dye - -d₁/dt rate of Ca²⁺ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - F_max (F_max — F_min)_cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F₅₀ fluorescence of the high-affinity form ofrhod2for[Ca²⁺]_cyt=50 nM - [Ca²⁺]₀ external Ca²⁺concentration - K _p proportionality constant between the total number of Ca²⁺ ions moved and the change in high-affinity rhod2 complexation to Ca² - (d[Ca²⁺]_{cyt, T})/dt rate of Ca²⁺ influx obtained with maximal levels of ionomycin - k_leak rate constant for passive inward Ca²⁺ leakage - k_inno rate constant for ionomycin-mediated Ca²⁺ influx - T total - [rhod2]_cyt,T total intracellular rhod2 concentration - [quin2]_cyt,T total intracellular quin2 concentration - [B]_T total cytoplasmic buffering capacity - A[Ca²⁺]_cyt,T total number of Ca²⁺ ions moved into the cytoplasm - [rhod2-Ca]_{cyt, T} change in concentration of total intracellular high-affinity rhod2 complexed to Ca²⁺ - [B-Ca]_T change in concentration of total cytoplasmic binding sites complexed to Ca²⁺ - [quin2]_{cyt, T} change in concentration of total intracellular quinl complexed to Ca²⁺ - change in the degree of intracellular quin2 saturation - ₁ change in degree of saturation of cytoplasmic high-affinity rhod2 - ₁-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - V_obs observed rate of Ca²⁺ removal from the rhod2-Ca complex - V_{8.3 m} the rate of Ca²⁺ removal from the high affinity rhod2-Ca complex at [Ca²⁺]_cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca²⁺]_cytT/_t initial linear rate of ionomycin-mediated Ca²⁺ influx - EC₅₀ effective concentration giving a half-maximal effect - [Na⁺]_cyt cytoplasmic Na⁺ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid     

Investigation of carvedilol-evoked Ca2+ movement and death in human oral cancer cells

The effect of carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca²⁺]_i in a concentration-dependent fashion. The Ca²⁺ signal was decreased by 50% by removing extracellular Ca²⁺. Carvedilol-induced Ca²⁺ entry was not affected by the store-operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin did not change carvedilol-induced [Ca²⁺]_i rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca²⁺ release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca²⁺]_i release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca²⁺]_i rise. Carvedilol at 5–50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca²⁺ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca²⁺]_i rise by causing phospholipase C-independent Ca²⁺ release from mitochondria and non-endoplasmic reticulum stores, and Ca²⁺ influx via protein kinase C-regulated channels. Carvedilol (up to 50 μM) induced cell death in a Ca²⁺-independent manner that involved apoptosis.     

Platelet-Activating Factor Evokes Ca2+ Transients After the Blockade of Ryanodine Receptor by Dantrolene in RAW 264.7 Macrophages

In the present study we studied platelet-activating factor (PAF)-, and ATP-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) using RAW 264.7 macrophages filled with fura-2/AM and imaged with fluorescence video microscopy. We found that the prevalence of detectable [Ca²⁺]_i responses to PAF application was significantly higher in the presence of dantrolene. Dantrolene itself significantly decreased basal [Ca²⁺]_i of macrophages compared to control cases after a 20-min incubation period. In the dantrolene-treated cells even the peak [Ca²⁺]_i in response to PAF (as an average of all cells) was below the baseline of control suggesting that decreased [Ca²⁺]_i plays a permissive role in the Ca²⁺ rise induced by PAF in macrophages. In contrast to the effect of PAF, neither the amplitude of response to ATP nor the frequency of responding cells changed significantly during dantrolene treatment in our experiments. These cells were able to respond to a standard immune stimulus as well: lipopolysaccharide (LPS) was able to increase [Ca²⁺]_i. Our data indicate that the effectiveness of PAF to increase [Ca²⁺]_i in RAW 264.7 macrophages depends on the resting [Ca²⁺]_i. It has also been shown in this study that PAF and ATP differently regulate Ca²⁺ homeostasis in macrophages during inflammatory response and therefore they possibly differently modulate cytokine production by macrophages.