Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca²⁺]_i, Ca²⁺ transients and membrane Ca²⁺ current, I_Ca, in cultured murine HL-1 cardiomyocytes. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca²⁺]_i, Ca²⁺ transients and I_Ca. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca²⁺]_i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca²⁺]_i, and inhibited Ca²⁺ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca²⁺]_i, and Ca²⁺ transients and I_Ca. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca²⁺]_i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca²⁺]_i required for excitation-contraction coupling in cardiomyoctyes.     

Oscillatory Cl current induced by angiotensin II in rat pulmonary arterial myocytes: Ca dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca²⁺]_i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca²⁺]_i. In cells clamped at −60 mV, ANG II (10 μM) or ATP (100 μM) induced an oscillatory inward current. Caffeine (5 μM) induced only one transient inward current. In control conditions, the reversal potential (E_rev) of these currents was close to the equilibrium potential for Cl⁻ ions (E_Cl = −2.1 mV) and was shifted towards more positive values in low-Cl⁻ solutions. Niflumic acid (10–50 μM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca²⁺]_i by Indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca²⁺]_i, whereas the caffeine-induced current was accompanied by only one transient increase in [Ca²⁺]_i Niflumic acid (25 μM) had no effect on agonist-induced [Ca²⁺]_i responses, whereas thapsigargin (1 μM) abolished both membrane current and the [Ca²⁺]_i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca²⁺]_i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 μM) or nifedipine (1 μM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca²⁺]_i and that both oscillatory phenomena are primarily IP₃ mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca²⁺ channels. The resulting Ca²⁺ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

Redox modulation of calcium entry and release of intracellular calcium by thimerosal in GH4C1 pituitary cells

In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in GH₄C₁ pituitary cells. In intact Fura-2 loaded cells, low concentrations of thimerosal potentiated the spike phase of the TRH-induced (thyrotropin-releasing hormone) rise in [Ca²⁺]_i, whereas high thimerosal concentrations inhibited it. The effect of thimerosal on the plateau phase was always inhibitory.The effect of thimerosal on the IP₃-induced calcium release (IICR) was studied in permeabilized cells using the Ca²⁺ indicator Fluo-3. A low concentration of thimerosal (10 μM) stimulated IICR: the Ca²⁺ release induced by 300 nM inositol-1,4,5-trisphosphate (IP₃) was enhanced in cells treated with thimerosal for 1 or 6 min (67 ± 11 nM and 34 ± 5 nM, respectively) as compared to control cells (17 ± 2 nM). On the other hand, a high concentration of thimerosal (100 μ inhibited IICR: when IP₃ (10 μM) was added after a 5 min preincubation with thimerosal, the IP₃-induced rise in [Ca²⁺]_i (46 ± 14 nM) was 57% smaller as compared with that seen in control cells (106 ± 10 nM).The effect of thimerosal on the voltage-operated Ca ²⁺ channels (VOCCs) was studied by depolarizing intact Fura-2 loaded cells by addition of 20 mM K⁺ to the cuvette. The depolarization-evoked increase in [Ca²⁺]_i was inhibited in a dose-dependent manner by thimerosal. Direct evidence for an inhibitory effect of thimerosal on VOCCs was obtained by using the whole-cell configuration of the patch-clamp technique: thimerosal (100 μM) potently inhibited the Ba²⁺ currents through VOCCs.In addition, our results indicated that thimerosal inhibited the caffeine-induced increase in [Ca²⁺]_i, and activated a capacitative Ca²⁺ entry pathway. The actions of thimerosal were apparently due to its oxidizing activity because the effects were mostly reversed by the thiol-reducing agent dithiothreitol (DTT).We conclude that, in GH₄C₁ pituitary cells, the mobilization of intracellular calcium and the different Ca²⁺ entry pathways are sensitive to redox modulation.     

Rise in cytoplasmic Ca induced by monensin in bovine medullary chromaffin cells

Monensin, a exchanger, induces catecholamine secretion from adrenal chromaffin cells by an unknown mechanism. We found and report here that in bovine chromaffin cells, monensin evokes profound changes in [Ca²⁺]_i which were measured by means of the fluorescent Ca²⁺ indicator Indo-1. Application of monensin (10 μM) generated a marked [Ca²⁺]_i rise. Removal of external Ca²⁺ did not prevent the elevation of [Ca²⁺]_i, though it was significantly decreased. In the presence of nifedipine (10 μM) or tetrodotoxin (3 μM) the monensin-induced [Ca²⁺]_i rise remained unchanged. In contrast, in the absence of extracellular Na⁺ the [Ca²⁺]_i rise was abolished. Addition of caffeine (40 mM) at the peak response generated by monensin produced a further increase in [Ca²⁺]_i, which was independent of external [Ca²⁺] or [Na⁺]. After depletion of the IP₃-sensitive compartment by thapsigargin (1 μM), caffeine still induced a rise in [Ca²⁺]_i while the monensin response was absent. We concluded that the origin of the Ca²⁺ for the [Ca²⁺]_i increase elicited by the exchanger in chromaffin cells is not the extracellular space. Clearly there seems to be at least two intracellular Ca²⁺ stores, one of which is affected by monensin. This Ca²⁺ pool, which is different than the pool stimulated by caffeine, is sensitive to the extracellular [Ca²⁺] and to thapsigargin. Our data are compatible with the idea that the monensin mediated Na⁺ entry could activate the production of inositol trisphosphate and this in turn could trigger Ca²⁺ release from the endoplasmic reticulum.     

Agonist-activated Ca2+ influx and Ca2+ -dependent Cl- channels in Xenopus ovarian follicular cells: functional heterogeneity within the cell monolayer

Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion‐current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage‐clamp technique. All agonists elicited Cl^? currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl^? (F_Cl) currents, while AII activated an oscillatory response; a robust Ca²⁺ influx linked specifically to F_Cl activation elicited an inward current (I_iw,Ca) which was carried mainly by Cl^? ions, through channels with a sequence of permeability of SCN^? > I^? > Br^? > Cl^?. Like F_Cl, I_iw,Ca was not dependent on oocyte [Ca²⁺]_i; instead both were eliminated by preventing [Ca²⁺]_i increase in the follicular cells, and also by U73122 and 2‐APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F_Cl and I_iw,Ca were produced by the expected, PLC‐stimulated Ca²⁺‐release and Ca²⁺‐influx, respectively, and by the opening of I_Cl(Ca) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca²⁺‐influx appeared to be driven through store‐operated, calcium‐like channels. The AII response, which is also known to require PLC activation, did not activate I_iw,Ca and was strictly dependent on oocyte [Ca²⁺]_i increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap‐junction channels. J. Cell. Physiol. 227: 3457–3470, 2012. © 2011 Wiley Periodicals, Inc.     

Passive Ca binding in ventricular myocardium of neonatal and adult rats

In this study, passive Ca²⁺ binding was determined in ventricular homogenates (VH) from neonatal (4–6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca²⁺ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca²⁺ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca²⁺] ([Ca²⁺]_F was measured with Indo-1 and bound Ca²⁺ ([Ca²⁺]_B) was the difference between [Ca²⁺]_F and total Ca²⁺. Apparent Ca²⁺ dissociation constants (K_d) for BAPTA and Indo-1 were increased by 10–20 mg VH protein/ml (from 0.35 to 0.92 μM for Indo-1 and from 0.20 to 0.76 μM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl₂ additions (2.5–10 nmoles) yielded δ[Ca²⁺]_B/δ[Ca²⁺]_F for the sum of [Ca²⁺]_B at all three classes of binding sites. From this function, the apparent number of endogenous sites (B_en) and their K_d (K_en) were determined. Similar K_en values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 ± 0.14 μM, 0.69 ± 0.13 μM and 0.53 ± 0.10 μM, respectively). However, B_en was significantly higher in adult myocytes than in adult VH (1.73 ± 0.35 versus 0.70 ± 0.12 nmol/mg protein, P < 0.01), which correspond to ∼300 and 213 μmol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that B_en determined in VH underestimates cellular B_en by 29%. Although B_en values in nmol/mg protein were similar in adult and neonatal VH (0.69 ± 0.12), protein content was much higher in adult ventricle (125 ± 7 versus 80 ± 1 mg protein/g wet weight, P < 0.01). Expressing B_en per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca²⁺ binding capacity at high-affinity sites is ∼300 and 176 mmol/I cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca²⁺ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month.     

Examination of the Effects of Heterogeneous Organization of RyR Clusters,Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes

Spatio-temporal dynamics of intracellular calcium, [Ca²⁺]_i, regulate the contractile function of cardiac muscle cells. Measuring [Ca²⁺]_i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca²⁺]_i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca²⁺ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca²⁺]_i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca²⁺]_i transient and of the simulated fluorescence intensity signal, F/F₀, reached values similar to that found in the literature ([Ca²⁺]_i ≈1 μM; F/F₀≈5.5). However, our model predicted the variation in [Ca²⁺]_i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F₀ signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca²⁺]_i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca²⁺]_i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca²⁺]_i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.     

Effect of caffeine on mucus secretion and agonist-dependent Ca2+ mobilization in human gastric mucus secreting cells

Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca²⁺]_i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca²⁺]_i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [³H]glucosamine. Caffeine by itself failed to increase [Ca²⁺]_i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca²+]_i rise, resulting in inhibiting activation of Ca²⁺-dependent K⁺ current (I_K.Ca) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca²⁺]_i rise and activation of I_K.Ca induced by A23187 or inositol trisphosphate (IP₃). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca²⁺]_i rise in human gastric epithelial cells, probably through the blockade of receptor-IP₃ signaling pathway, which may affect the mucin secretion. © 1997 Elsevier Science B.V. All rights reserved.     

Regulation of store-operated Ca entry in pulmonary artery smooth muscle cells

Store-operated Ca²⁺ entry (SOCE) is an important mechanism for Ca²⁺ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca²⁺-insensitive phospholipase A₂ (iPLA₂) is involved in the activation of SOCE in many different cell types. The iPLA₂ inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca²⁺ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca²⁺ influx was blocked by Gd³⁺ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway.     

Activation of a capacitative Ca entry pathway by store depletion in cultured hippocampal neurones

Intracellular Ca²⁺ ([Ca²⁺]_i) changes were measured in cell bodies of cultured rat hippocampal neurones with the fluorescent indicator Fluo-3. In the absence of external Ca²⁺, the cholinergic agonist carbachol (200 μM) and the sarco-endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (0.4 μM) both transiently elevated [Ca²⁺]_i. A subsequent addition of Ca²⁺ into the bathing medium caused a second [Ca²⁺]_i change which was blocked by lanthanum (50 μM). Taken together, these experiments indicate that stores depletion can activate a capacitative Ca²⁺ entry pathway in cultured hippocampal neurones and further demonstrate the existence of such a Ca²⁺ entry in excitable cells.     

Voltage-Induced Ca2+ Release in Postganglionic Sympathetic Neurons in Adult Mice

Recent studies have provided evidence that depolarization in the absence of extracellular Ca²⁺ can trigger Ca²⁺ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca²⁺ from intracellular stores in response to depolarization, independent of Ca²⁺ influx. We measured changes in cytosolic ΔF/F₀ in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K⁺ depolarization in the presence (2 mM) and absence of extracellular Ca²⁺ ([Ca²⁺]_e). Progressive elevations in extracellular [K⁺]_e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca²⁺]_e. Peak amplitude of ΔF/F₀ transients in 2 mM [Ca²⁺]_e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F₀ in 0 mM [Ca²⁺]_e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca²⁺]_e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F₀ transients in 0 mM [Ca²⁺]_e were slower than those of ΔF/F₀ transients evoked in 2 mM [Ca²⁺]_e. Rises in ΔF/F₀ evoked by high [K⁺]_e in the absence of extracellular Ca²⁺ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ ATPase, or the inositol 1,4,5-triphosphate (IP₃) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd²⁺, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca²⁺ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca²⁺ from IP₃-sensitive internal stores in response to membrane depolarization, independent of Ca²⁺ influx.     

In Intact Cone Photoreceptors,a Ca2+-dependent,Diffusible Factor Modulates the cGMP-gated Ion Channels Differently than in Rods

We investigated the modulation of cGMP-gated ion channels in single cone photoreceptors isolated from a fish retina. A new method allowed us to record currents from an intact outer segment while controlling its cytoplasmic composition by superfusion of the electropermeabilized inner segment. The sensitivity of the channels to agonists in the intact outer segment differs from that measured in membrane patches detached from the same cell. This sensitivity, measured as the ligand concentration necessary to activate half-maximal currents, K _1/2, also increases as Ca²⁺ concentration decreases. In electropermeabilized cones, K _1/2 for cGMP is 335.5 ± 64.4 μM in the presence of 20 μM Ca²⁺, and 84.3 ± 12.6 μM in its absence. For 8Br-cGMP, K _1/2 is 72.7 ± 11.3 μM in the presence of 20 μM Ca²⁺ and 15.3 ± 4.5 μM in its absence. The Ca²⁺-dependent change in agonist sensitivity is larger in extent than that measured in rods. In electropermeabilized tiger salamander rods, K _1/2 for 8Br-cGMP is 17.9 ± 3.8 μM in the presence of 20 μM Ca²⁺ and 7.2 ± 1.2 μM in its absence. The Ca²⁺-dependent modulation is reversible in intact cone outer segments, but is progressively lost in the absence of divalent cations, suggesting that it is mediated by a diffusible factor. Comparison of data in intact cells and detached membrane fragments from cones indicates that this factor is not calmodulin. At 40 μM 8Br-cGMP, the Ca²⁺-dependent change in sensitivity in cones is half-maximal at K _Ca = 286 ± 66 nM Ca²⁺. In rods, by contrast, K _Ca is ∼50 nM Ca²⁺. The difference in magnitude and Ca²⁺ dependence of channel modulation between photoreceptor types suggests that this modulation may play a more significant role in the regulation of photocurrent gain in cones than in rods.     

The role of cytoplasmic [Ca] in glucose-induced inhibition of respiration and oxidative phosphorylation in Ehrlich ascites tumour cells: a novel mechanism of the Crabtree effect

The effect of Ca²⁺ on energy-coupling parameters of Ehrlich ascites carcinoma was studied in digitonin-permeabilized cells. In nominally Ca-free medium the permeabilized cells respond to the addition of ADP by increased oxygen uptake with externally added respiratory substrates (succinate or pyruvate), decrease of the mitochondrial membrane potential (Δψ) and alkalinization of the medium. This typical behaviour is drastically changed if Ca²⁺ is added. The subsequent addition of ADP induces neither State 3 respiration, nor decrease of Δψ, nor alkalinization of the medium, indicating a complete block of ATP synthesis. These effects are produced by both a single pulse of 100 μM Ca²⁺ and a preincubation for 2 min with 0.4–1.0 μM Ca²⁺. Preincubation of the cells with glucose or deoxyglucose prior to permeabilization makes them sensitive to Ca²⁺ concentrations as low as 0.3 μM. In view of the previous finding that glucose and deoxyglucose produce an increase of cytoplasmic [Ca²⁺] in Ehrlich ascites cells [Teplova VV. Bogucka K. Czyż A. Evtodienko YuV. Duszyński J. Wojtczak L. (1993) Biochem. Biophys. Res. Commun., 196, 1148–1154; Czyż A. Teplova VV. Sabała P. Czarny M. Evtodienko YuV. Wojtczak L. (1993) Acta Biochim. Polon., 40, 539–544], the present results suggest that cytoplasmic Ca²⁺ plays a crucial role in the Crabtree effect.     

Generation of repetitive Ca transients by bombesin requires intracellular release and influx of Ca through voltage-dependent and voltage independent channels in single HIT cells

In the present study, the bombesin-induced changes in cytosolic free Ca²⁺ ([Ca²⁺]_i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca²⁺ transients. The average frequency of the Ca²⁺ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min⁻¹ (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca²⁺ transient followed by a sustained plateau or by a decrease to basal levels. In Ca²⁺- free medium, bombesin caused only one or two Ca²⁺ transients and withdrawal of extracellular Ca²⁺ abolished the Ca²⁺ transients. The voltage-dependent Ca²⁺ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca²⁺ transients and stopped the Ca²⁺ transients in some cells. Thapsigargin caused a sustained rise in [Ca²⁺]_i) in the presence of extracellular Ca²⁺ while in its absence the rise in [Ca²⁺]_i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca²⁺], by about 50%. Depletion of intracellular Ca²⁺ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca²⁺-free medium caused an agonist-independent increase in [Ca²⁺]_i) when extracellular Ca²⁺ was restored, which was larger than in control cells that had been incubated in Ca²⁺-free medium for the same period of time. This rise in [Ca²⁺]_i and the thapsigargin-induced increase in [Ca²⁺]_i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca²⁺ pool enhances Ca²⁺ influx through VDCC and voltage-independent Ca²⁺ channels (VICC). In conclusion, the bombesin-induced Ca²⁺ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca²⁺ transients. Intracellular Ca²⁺ is mobilized during each Ca²⁺ transient, but Ca²⁺ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca²⁺ response. Ca²⁺ influx in whole or part is activated by a capacitative Ca²⁺ entry mechanism.     

Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.     

Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (_ΔΨ_m), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]_m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²⁺]_m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²⁺ indicators with a 20-fold difference in Ca²⁺ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²⁺ indicator (D4cpv) calibrated against known Ca²⁺ levels. Matrix pH (pH_m) and _ΔΨ_m were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²⁺]_m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²⁺]_m coincided with a rapid elevation in pH_m and was followed by an after-stimulus _ΔΨ_m hyperpolarization. However, pH_m decreased and no _ΔΨ_m hyperpolarization was observed in response to lower levels of [Ca²⁺]_m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca²⁺]_m are required to stimulate presynaptic mitochondrial energy metabolism.     

The peripheral Ca pump activity of macrophages and neutrophils

Exposure of either alveolar macrophages or blood neutrophils to 0.2 – 1 μM ionophore A23187 in the presence of 0.1 – 1 mM CaCl₂ causes a rapid extracellular release of Ca²⁺, which can be measured by a Ca²⁺-selective electrode. The initial rate at which the cation is extruded from the cells is about 0.1 – 0.2 μg-ions/min/ml of cell water. ATP depletion, but not replacement of extracellular Na⁺ with choline, produces a marked inhibition of Ca²⁺ release from macrophages. When the movements of Ca²⁺ between neutrophils and the incubation medium are followed by an isotopic technique, a transient increase in cell-associated ⁴⁵Ca²⁺ is detected a few seconds after the addition of the ionophore. We suggest that the ionophore A23187 mobilises Ca²⁺ from intracellular stores, with a subsequent cell extrusion of the bivalent cation catalysed by a pump localised at the cell surface. These and other data are consistent with the conclusion that the peripheral Ca²⁺ pump system of macrophages and neutrophils is very similar to the well know Ca²⁺ pump of the red cells with regard to mechanism and capacity.     

Dysfunction of mitochondria Ca uptake in cystic fibrosis airway epithelial cells

In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca²⁺ mobilization is abnormally increased. Because the uptake of Ca²⁺ by mitochondria during Ca²⁺ influx or Ca²⁺ release from ER stores may be crucial for maintaining a normal Ca²⁺ homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (ΔΨ_mit) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2–AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca²⁺. The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the ΔΨ_mit of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca²⁺ uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca²⁺ uptake.     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.