Distinct Mechanisms of [Ca2+]i Oscillations in HSY and HSG Cells: Role of Ca2+ Influx and Internal Ca2+ Store Recycling

This study examined [Ca²⁺]_i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca²⁺]_i and K _Ca currents in both cell types. Low IP₃, but not thapsigargin (Tg), induced [Ca²⁺]_i oscillations, whereas Tg blocked CCh-stimulated [Ca²⁺]_i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca²⁺ from HSY cells (i) did not affect CCh-stimulated [Ca²⁺]_i oscillations or internal Ca²⁺ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca²⁺]_i into oscillations. CCh- or thapsigargin-induced Ca²⁺ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic reticulum Ca²⁺ pump (SERCA) and inositoltrisphosphate receptors (IP₃Rs) than HSG cells. These data demonstrate that [Ca²⁺]_i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca²⁺ from, and release of Ca²⁺ into, the cytosol by the SERCA and IP₃R activities, respectively. In HSY cells, Ca²⁺ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca²⁺]_i. In HSG cells, [Ca²⁺]_i oscillations directly depend on Ca²⁺ influx; Ca²⁺ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences in the mechanism of [Ca²⁺]_i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca²⁺ stores. Received: 30 October 2000/Revised: 26 February 2001     

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.     

Caffeine-induced Ca2+ oscillations in type I horizontal cell of carp retina: A mathematical model

Oscillations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) have been observed in a variety of cell types. In the present study, we constructed a mathematical model to simulate the caffeine-induced [Ca²⁺]_i oscillations based on experimental data obtained from isolated type I horizontal cell of carp retina. The results of model analysis confirm the notion that the caffeine-induced [Ca²⁺]_i oscillations involve a number of cytoplasmic and endoplasmic Ca²⁺ processes that interact with each other. Using this model, we evaluated the importance of store-operated channel (SOC) in caffeine-induced [Ca²⁺]_i oscillations. The model suggests that store-operated Ca²⁺ entry (SOCE) is elicited upon depletion of the endoplasmic reticulum (ER). When the SOC conductance is set to 0, caffeine-induced [Ca²⁺]_i oscillations are abolished, which agrees with the experimental observation that [Ca²⁺]_i oscillations were abolished when SOC was blocked pharmacologically, verifying that SOC is necessary for sustained [Ca²⁺]_i oscillations.     

Caffeine-induced Ca2+ oscillations in type I horizontal cell of carp retina: A mathematical model

Oscillations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) have been observed in a variety of cell types. In the present study, we constructed a mathematical model to simulate the caffeine-induced [Ca²⁺]_i oscillations based on experimental data obtained from isolated type I horizontal cell of carp retina. The results of model analysis confirm the notion that the caffeine-induced [Ca²⁺]_i oscillations involve a number of cytoplasmic and endoplasmic Ca²⁺ processes that interact with each other. Using this model, we evaluated the importance of store-operated channel (SOC) in caffeine-induced [Ca²⁺]_i oscillations. The model suggests that store-operated Ca²⁺ entry (SOCE) is elicited upon depletion of the endoplasmic reticulum (ER). When the SOC conductance is set to 0, caffeine-induced [Ca²⁺]_i oscillations are abolished, which agrees with the experimental observation that [Ca²⁺]_i oscillations were abolished when SOC was blocked pharmacologically, verifying that SOC is necessary for sustained [Ca²⁺]_i oscillations.     

The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices

Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca²⁺]_i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca²⁺]_i change with initial transient phase followed by a plateau phase with highly synchronized [Ca²⁺]_i oscillations. Here, we aimed to correlate the plateau [Ca²⁺]_i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca²⁺]_i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca²⁺]_i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca²⁺]_i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca²⁺]_i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network.     

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.     

STIM1 Regulates Platelet-Derived Growth Factor-Induced Migration and Ca2+ Influx in Human Airway Smooth Muscle Cells

It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca²⁺ sensor that activates Orai1, the Ca²⁺ channel responsible for store-operated Ca²⁺ entry (SOCE). We investigated the role of STIM1 in [Ca²⁺]_i and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca²⁺-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca²⁺]_i followed by sustained [Ca²⁺]_i elevation. Sustained increases in [Ca²⁺]_i due to PDGF-BB were significantly inhibited by a Ca²⁺ chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca²⁺]_i or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca²⁺ influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling.     

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.     

Muscarinic Activation of BK Channels Induces Membrane Oscillations in Glioma Cells and Leads to Inhibition of Cell Migration

Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca²⁺] _i ) associated with periodic Ca²⁺ oscillations during prolonged ACh applications. The early transient rise in [Ca²⁺] _i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca²⁺ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca²⁺] _i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca²⁺-sensitive K⁺ currents. These K⁺ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca²⁺ from intracellular stores which in turn activate Ca²⁺-dependent (BK-type) K⁺ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration. Received: 17 December/Revised: 17 March 2000     

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.     

Tachyphylaxis to the inhibitory effect of L-type channel blockers on ACh-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> oscillations in porcine tracheal myocytes

Summary Discrepancies about the role of L-type voltage-gated calcium channels (VGCC) in acetylcholine (ACh)-induced [Ca²⁺]_i oscillations in tracheal smooth muscle cells (TSMCs) have been seen in recent reports. We demonstrate here that ACh-induced [Ca²⁺]_i oscillations in TMCS were reversibly inhibited by three VGCC blockers, nicardipine, nifedipine and verapamil. Prolonged (several minutes) application of VGCC blockers, led to tachyphylaxis; that is, [Ca²⁺]_i oscillations resumed, but at a lower frequency. Brief (15–30 s) removal of VGCC blockers re-sensitized [Ca²⁺]_i oscillations to inhibition by the agents. Calcium oscillations tolerant to VGCC blockers were abolished by KB-R7943, an inhibitor of the reverse mode of Na⁺/Ca²⁺ exchanger (NCX). KB-R7943 alone also abolished ACh-induced [Ca²⁺]_i oscillations. Enhancement of the reverse mode of NCX via removing extracellular Na⁺ reversed inhibition of ACh-induced [Ca²⁺]_i oscillations by VGCC blockers. Inhibition of non-selective cation channels using Gd³⁺ slightly reduced the frequency of ACh-induced [Ca²⁺]_i oscillations, but did not prevent the occurrence of tachyphylaxis. Altogether, these results suggest that VGCC and the reverse mode of NCX are two primary Ca²⁺ entry pathways for maintaining ACh-induced [Ca²⁺]_i oscillations in TSMCs. The two pathways complement each other, and may account for tachyphylaxis of ACh-induced [Ca²⁺]_i oscillations to VGCC blockers.     

Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion

Cyclic AMP (cAMP) and Ca²⁺ are key regulators of exocytosis in many cells, including insulin-secreting β cells. Glucose-stimulated insulin secretion from β cells is pulsatile and involves oscillations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse β cells. These oscillations were preceded and enhanced by elevations of [Ca²⁺]_i. However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca²⁺]_i rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.     

Mitochondrial membrane potential (DeltaPsi) and Ca<Superscript>2+</Superscript>-induced differentiation in HaCaT keratinocytes

We have used the human calcium- and temperature-dependent (HaCaT) keratinocyte cell line to elucidate mechanisms of switching from a proliferating to a differentiating state. When grown in low calcium medium (<0.1 mM) HaCaT cells proliferate. However, an increase in the calcium concentration of the culture medium, [Ca²⁺]₀, induces growth arrest and the cells start to differentiate. Numerous studies have already shown that the increase in [Ca²⁺]₀ results in acute and sustained increases in intracellular calcium concentration, [Ca²⁺]_i. We find that the Ca²⁺-induced cell differentiation of HaCaT cells is also accompanied by a significant decrease in mitochondrial membrane potential, DeltaPsi. By combining patch-clamp electrophysiological recordings and microspectrofluorimetric measurements of DeltaPsi on single cells, we show that the increase in [Ca²⁺]_i led to DeltaPsi depolarization. In addition, we report that tetraethylammonium (TEA), a blocker of plasma membrane K⁺ channels, which is known to inhibit cell proliferation, and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), a blocker of plasma membrane Cl^– channels, also affect DeltaPsi. Both these agents stimulate HaCaT cell differentiation. These data therefore strongly suggest a direct causal relationship between depolarization of DeltaPsi and the inhibition of proliferation and induction of differentiation in HaCaT keratinocytes.     

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.     

Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N‐terminal peptide (NT‐S100A8)

After isolating NT‐S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca²⁺]_i oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT‐S100A8. In NT‐S100A8 stimulated β‐TC6 (insulinoma cell line) culture medium, insulin and [Ca²⁺] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca²⁺]_i oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT‐S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT‐S100A8 induced a rapid insulin release and enhanced β‐TC6 [Ca²⁺]_i oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT‐S100A8, [Ca²⁺] in β‐TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT‐S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB‐α, but it independently activated Akt and NF‐κB signaling in PC cells. In conclusion, NT‐S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF‐κB signaling, NT‐S100A8 enhances [Ca²⁺]_i oscillations and insulin release, probably by inducing Ca²⁺ influx from the extracellular space, but it does not interfere with insulin signaling. J. Cell. Physiol. 226: 456–468, 2011. © 2010 Wiley‐Liss, Inc.     

Inositol 1,4,5‐trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca²⁺]_i), which is almost entirely mediated by inositol 1,4,5‐trisphosphate receptor 1 (IP₃R1). In mammalian eggs, fertilization‐induced [Ca²⁺]_i responses exhibit a periodic pattern that are called [Ca²⁺]_i oscillations. These [Ca²⁺]_i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP₃R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP₃R1 degradation and examined the impact of the IP₃R1 levels on the pattern of [Ca²⁺]_i oscillations. Using microinjection of IP₃ and of its analogs and conditions that prevent the development of [Ca²⁺]_i oscillations, we show that IP₃R1 degradation requires uniform and persistently elevated levels of IP₃. We also established that progressive degradation of the IP₃R1 results in [Ca²⁺]_i oscillations with diminished periodicity while a near complete depletion of IP₃R1s precludes the initiation of [Ca²⁺]_i oscillations. These results provide insights into the mechanism involved in the generation of [Ca²⁺]_i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley‐Liss, Inc.     

Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca2+]i Signals Determine Specificity of Ca2+-Dependent Gene Expression

Regulation of critical cellular functions, including Ca²⁺-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca²⁺ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca²⁺]_i due to intracellular Ca²⁺ release and Ca²⁺ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca²⁺]_i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca²⁺]_i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca²⁺ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca²⁺ store depletion in these cells, it is not clear how the cells decode individual Ca²⁺ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca²⁺]_i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca²⁺ entry generates [Ca²⁺]_i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca²⁺ entry generates sustained [Ca²⁺]_i elevation at high [CCh] and contributes to frequency of [Ca²⁺]_i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca²⁺ dependent gene expression pathways, consistent with the different patterns of [Ca²⁺]_i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca²⁺]_i generated by Orai1, independent of global [Ca²⁺]_i changes or TRPC1-mediated Ca²⁺ entry. In contrast, Ca²⁺ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca²⁺ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca²⁺-dependent gene expression pathways.     

Involvement of mitochondria in intracellular calcium sequestration by rat gonadotropes

Intracellular Ca²⁺ ([Ca²⁺]_i) dynamics were studied in identified rat gonadotropes using the whole-cell patch-clamp technique in conjunction with Indo-1 photometry. The kinetics of depolarization-induced [Ca²⁺]_i transients vary with Ca²⁺ load. In addition to a rapid initial decay, large (> 500 nM) [Ca²⁺]_i transients have a slow plateau phase. Application of the mitochondrial inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly slows the decay of [Ca²⁺]_i transients, consistent with stopping uptake of Ca²⁺ by mitochondria. CCCP causes a small increase of [Ca²⁺]_i at rest. After a large Ca²⁺ entry the amount is much larger, consistent with release from a mitochondrial Ca²⁺ pool that fills during cytoplasmic Ca²⁺ loading. The rate of Ca²⁺ uptake by mitochondria is dependent upon [Ca ²⁺]_i. Consistent with previous studies, gonadotropin releasing hormone (GnRH) induces [Ca²⁺]_i oscillations. The mitochondrial inhibitors CCCP and cyanide (CN⁻) terminate these oscillations. The mitochondrial ATP-synthase inhibitor oligomycin reduces the frequency and increases the amplitude of the oscillations. In the presence of ruthenium red (a non-specific blocker of the mitochondrial Ca²⁺-uniporter) in the pipette, GnRH does not induce rhythmic [Ca²⁺]_i oscillations. We suggest that mitochondria play a significant role in the rapid clearance of cytosolic Ca²⁺ loads in gonadotropes and participate in GnRH-induced periodic [Ca²⁺]_i oscillations.