Local Ca2+ signals in cellular signalling

Local Ca2+ rises and propagated Ca2+ signals represent different patterns that are differentially decoded for fine tuning cellular signalling. This Ca2+ concentration plasticity is absolutely required to allow adaptation to different needs of the cells ranging from contraction or increased learning to proliferation and cell death. A wide diversity of molecular structures and specific location of Ca2+ signalling molecules confer spatial and temporal versatility to the Ca2+ changes allowing specific cellular responses to be elicited. Various types of local Ca2+ signals have been described. Ca2+ spikes correspond to Ca2+ signals spanning several micrometers but displaying limited propagation into a cell leading to regulation of cellular functions in one particular zone of this cell. This is of particular relevance in cells presenting distinct morphological specializations, i.e. apical versus basal sites or dendritic versus somatic/axonal sites. More stereotyped elementary Ca2+ events (denominated Ca2+ sparks or Ca2+ puffs depending on the type of endoplasmic reticulum Ca2+ release channel involved) are highly confined and non-propagated Ca2+ rises which are observed in the close neighbouring of the Ca2+ channels. These elementary Ca2+ events play a major role in controlling cellular excitability. Elementary Ca2+ events involve Ca2+ release channels such as the ryanodine receptors (RyRs) and the inositol 1,4,5-trisphosphate receptors (InsP3Rs). The molecular bases underlying the various local Ca2+ release events will be discussed by reviewing the channels and particularly the different isoforms of RyRs and InsP3Rs and their role in inducing localized Ca2+ responses. These calcium release events are controlled by various second messengers and are regulated by Ca2+ channel-associated proteins, intra-luminal Ca2+ content of the endoplasmic reticulum (ER) and other Ca2+ organelles. We will discuss on how the control of local cellular Ca2+ content may account for cellular functions in physiological and physiopathological conditions.     

Extracellular Ca²+ alleviates NaCl-induced stomatal opening through a pathway involving H₂O₂-blocked Na+ influx in Vicia guard cells

To gain further insights into the function of extracellular Ca2+ in alleviating salt stress, Vicia faba guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that 100 mM NaCl clearly induced Na+ influx across the plasma membrane in GCPs and promoted stomatal opening. Extracellular Ca2+ at 10 mM efficiently blocked Na+ influx and inhibited stomatal opening, which was partially abolished by La3+ (an inhibitor of plasma membrane Ca2+ channel) or catalase (CAT, a H?O? scavenger), respectively. These results suggest that the plasma membrane Ca2+ channels and H?O? possibly mediate extracellular Ca2+-blocked Na+ influx in GCPs. Furthermore, extracellular Ca2+ activated the plasma membrane Ca2+ channels under NaCl stress, which was partially abolished by CAT. These results, taken together, indicate that hydrogen peroxide (H?O?) likely regulates Na+ uptake by activating plasma membrane Ca2+ channels in GCPs. In accordance with this hypothesis, H?O? could mimic extracellular Ca2+ to activate Ca2+ channels and block Na+ influx in guard cells. A single-cell analysis of cytosolic free Ca2+ ([Ca2+](cyt)) using Fluo 3-AM revealed that extracellular Ca2+ induced the accumulation of cytosolic Ca2+ under NaCl stress, but had few effects on the accumulation of cytosolic Ca2+ under non-NaCl conditions. All of these results, together with our previous studies showing that extracellular Ca2+ induced the generation of H?O? in GCPs during NaCl stress, indicate that extracellular Ca2+ alleviates salt stress, likely by activating the H?O?-dependent plasma membrane Ca2+ channels, and the increase in cytosolic Ca2+ appears to block Na+ influx across the plasma membrane in Vicia guard cells, leading to stomatal closure and reduction of water loss.     

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.     

Regulatory changes in the K+, Cl- and water contents of HeLa cells incubated in an isosmotic high K(+)-medium

HeLa cells had their normal medium replaced by an isosmotic medium containing 80 mM K+, 70 mM Na+ and 100 microM ouabain. The cellular contents of K+ first increased and then decreased to the original values, that is, the cells showed a regulatory decrease (RVD) in size. The initial increase was not inhibited by various agents except by substitution of medium Cl- with gluconate. In contrast, the regulatory decrease was inhibited strongly by addition of either 1 mM quinine, 10 microM BAPTA-AM without medium Ca2+, or 0.5 mM DIDS, and partly by either 1 mM EGTA without medium Ca2+, 10 microM trifluoperazine, or substitution of medium Cl- with NO3-. Addition of DIDS to the NO3(-)-substituted medium further suppressed the K+ loss but the effect was incomplete. Intracellular Ca2+ showed a transient increase after the medium replacement. These results suggest that the initial increase in cell K+ is a phenomenon related to osmotic water movement toward Donnan equilibrium, whereas the regulatory K+ decrease is caused by K+ efflux through Ca(2+)-dependent K+ channels. The K+ decrease induced a decrease in cellular water, i.e., RVD. The K+ efflux may be more selectively associated with Cl- efflux through DIDS-sensitive channels than the efflux of other anions.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Different contributions of voltage-sensitive Ca2+ channels to histamine-induced catecholamine release and tyrosine hydroxylase activation in bovine adrenal chromaffin cells.

Histamine stimulates catecholamine release and tyrosine hydroxylase activity in a Ca(2+)-dependent manner in bovine adrenal chromaffin cells. The role of voltage-sensitive Ca2+ channels in these two responses has been investigated. Using an EC50 concentration of histamine, 1 microM, catecholamine release was enhanced by (+/-)BayK8644, and partially inhibited by nitrendipine and omega-agatoxin IVA, blockers of L- and P/Q-type Ca2+ channels. omega-Conotoxin GVIA gave small and variable inhibitory effects. With a maximal histamine concentration, 10 microM, similar results were obtained except that now omega-conotoxin GVIA reliably reduced release. In contrast, neither (+/-)BayK8644 nor any of the individual Ca2+ channel antagonists had any significant effect on tyrosine hydroxylase (TOH) activation induced by either an EC50 or a maximal concentration of histamine. When high concentrations of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA were combined with omega-conotoxin MVIIC (a non-selective blocker of N, P and Q channels) to block voltage-sensitive Ca2+ channels in these cells, release induced by K+ depolarization was completely blocked. Release caused by histamine, however, was substantially reduced but not abolished. The combination of antagonists also only partially inhibited TOH activation by histamine. The results show that the G protein-coupled receptor agonist histamine activates several different types of voltage-sensitive Ca2+ channels in chromaffin cells to mediate its cellular effects. Histamine may also activate additional pathways for Ca2+ entry. The results also suggest that the manner by which Ca2+ controls release and TOH activation once it has entered chromaffin cells through these channels are different.     

Ionic events induced by epidermal growth factor. Evidence that hyperpolarization and stimulated cation influx play a role in the stimulation of cell growth.

Charybdotoxin, a blocker of K+ channels, and the imidazole drug SC38249, a blocker of both voltage- and second messenger-operated Ca2+ channels, were employed in mouse NIH-3T3 fibroblasts overexpressing the epidermal growth factor (EGF) receptor 1) to characterize the ionic events activated by EGF; and 2) to establish the role of those events in cell growth. The [Ca2+]i response by EGF was little changed by charybdotoxin while the parallel hyperpolarization was inhibited in a dose-dependent manner. At high toxin concentrations (greater than 3 x 10(-8) M), the effect of EGF on membrane potential was turned into a persistent depolarization sustained by both Na+ and Ca2+. Pretreatment with 10 microM SC38249 induced only minor changes of the intracellular Ca2+ release by EGF (the process responsible for the initial phase of the [Ca2+]i and membrane potential responses) and blocked the persistent, second phase [Ca2+]i and the hyperpolarization responses, both dependent on Ca2+ influx, as well as the depolarization in the charybdotoxin-pretreated cells. Long term (up to 2-day) treatment with either charybdotoxin or SC38249 failed to affect the viability and growth of unstimulated EGFR-T17 cells. Moreover, in these cells, the ionic responses to EGF were restored after a 30-min incubation in fresh medium. In contrast, growth stimulated by EGF was inhibited, moderately (-20%) by charybdotoxin and markedly (-60%) by SC38249. These results indicate for the first time that both hyperpolarization and, especially, the persistent increase of [Ca2+]i sustained by Ca2+ influx play a role in the activity of EGF, ultimately cooperating with other intracellular events in mitogenesis.     

Maitotoxin potently promotes Ca2+ influx in mouse spermatogenic cells and sperm, and induces the acrosome reaction

Maitotoxin (MTX), a potent marine toxin, activates Ca2+ entry via nonselective cation channels in a wide variety of cells. The identity of the channels involved in MTX action remains unknown. In mammalian sperm, Ca2+ entry through store-operated channels regulates a number of physiological events including the acrosome reaction (AR). Here we report that MTX produced an increase in the intracellular concentration of Ca2+ ([Ca2+]i) in spermatogenic cells that depended on extracellular Ca2+. Ni2+ and SKF96365 diminished the MTX-activated Ca2+ uptake, at concentrations they inhibit store-operated channels, and in a similar manner as they inhibit the Ca2+ influx activated following depletion of intracellular stores by thapsigargin (Tpg). In addition, MTX significantly increased [Ca2+]i in single mature sperm and effectively induced the AR with a half-maximal concentration (ED50) of approximately 1.1 nM. Notably, SKF96365 similarly inhibited the MTX-induced increase in sperm [Ca2+]i and the AR triggered by the toxin, Tpg and zona pellucida. These results suggest that putative MTX-activated channels may be involved in the Ca2+ influx required for mouse sperm AR.     

AMP-activated protein kinase mediates carotid body excitation by hypoxia

Early detection of an O2 deficit in the bloodstream is essential to initiate corrective changes in the breathing pattern of mammals. Carotid bodies serve an essential role in this respect; their type I cells depolarize when O2 levels fall, causing voltage-gated Ca2+ entry. Subsequent neurosecretion elicits increased afferent chemosensory fiber discharge to induce appropriate changes in respiratory function (1). Although depolarization of type I cells by hypoxia is known to arise from K+ channel inhibition, the identity of the signaling pathway has been contested, and the coupling mechanism is unknown (2). We tested the hypothesis that AMP-activated protein kinase (AMPK) is the effector of hypoxic chemotransduction. AMPK is co-localized at the plasma membrane of type I cells with O2-sensitive K+ channels. In isolated type I cells, activation of AMPK using 5-aminoimidazole-4-carboxamide riboside (AICAR) inhibited O2-sensitive K+ currents (carried by large conductance Ca2+-activated (BKCa) channels and TASK (tandem pore, acid-sensing potassium channel)-like channels, leading to plasma membrane depolarization, Ca2+ influx, and increased chemosensory fiber discharge. Conversely, the AMPK antagonist compound C reversed the effects of hypoxia and AICAR on type I cell and carotid body activation. These results suggest that AMPK activation is both sufficient and necessary for the effects of hypoxia. Furthermore, AMPK activation inhibited currents carried by recombinant BKCa channels, whereas purified AMPK phosphorylated thealpha subunit of the channel in immunoprecipitates, an effect that was stimulated by AMP and inhibited by compound C. Our findings demonstrate a central role for AMPK in stimulus-response coupling by hypoxia and identify for the first time a link between metabolic stress and ion channel regulation in an O2-sensing system.     

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

Inhibition of inositol phosphate production is a late, Ca2+-dependent effect of D2 dopaminergic receptor activation in rat lactotroph cells

The effect of dopamine, working through the activation of D2 receptors, on inositol phosphate production induced by thyrotropin-releasing hormone (TRH) was investigated in rat pituitary lactotroph cells. Dopamine (10 microM) did not modify the initial rapid stimulation of inositol 1,4,5-triphosphate and inositol bisphosphate observed within the first 15 s after TRH addition, but progressively inhibited the later inositol phosphate production induced by the neurohormone. This kinetics of inhibition was independent of dopamine preincubation time (from 2 to 10 min). The effect was still visible when dopamine was added after TRH. It was sensitive to pertussis toxin, was unchanged by increasing cellular cAMP levels with 8-Br-cAMP, but was greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the dopamine-induced inhibition was prevented by treatment of the cells with the Ca2+ ionophore ionomycin (100-200 nM) and was mimicked either by withdrawal of Ca2+ from the incubation medium or by blockade of voltage-gated Ca2+ channels with verapamil. The dopamine treatment did not decrease the cellular levels of the various phosphoinositides, strongly suggesting that the inhibition of inositol phosphate production is not due to precursor depletion. In isolated membranes, however, dopamine was unable to counteract the inositol phosphate accumulation triggered by TRH. Taken together, the data indicate that inhibition of inositol phosphate production is not a primary event triggered by D2 receptor activation, but is a late consequence, due to the previously demonstrated (Malgaroli, A., Vallar, L., Reza Elahi, F., Pozzan, T., Spada, A., and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927) inhibition by dopamine of the prolonged cytosolic free Ca2+ concentration increase induced by TRH via the activation of voltage-gated Ca2+ channels. These results are inconsistent with the possibility of a direct inhibitory coupling of D2 receptors to phospholipase C in rat pituitary lactotroph cells.     

Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).

We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel.     

Oxygen sensing by ion channels and chemotransduction in single glomus cells

We have monitored cytosolic [Ca2+] and dopamine release in intact fura- 2-loaded glomus cells with microfluoroimetry and a polarized carbon fiber electrode. Exposure to low PO2 produced a rise of cytosolic [Ca2+] with two distinguishable phases: an initial period (with PO2 values between 150 and approximately 70 mm Hg) during which the increase of [Ca2+] is very small and never exceeds 150-200 nM, and a second phase (with PO2 below approximately 70 mm Hg) characterized by a sharp rise of cytosolic [Ca2+]. Secretion occurs once cytosolic [Ca2+] reaches a threshold value of 180 +/- 43 nM. The results demonstrate a characteristic relationship between PO2 and transmitter secretion at the cellular level that is comparable with the relation described for the input (O2 tension)output (afferent neural discharges) variables in the carotid body. Thus, the properties of single glomus cells can explain the sensory functions of the entire organ. In whole-cell, patch- clamped cells, we have found that in addition to O2-sensitive K+ channels, there are Ca2+ channels whose activity is also regulated by PO2. Ca2+ channel activity is inhibited by hpoxia, although in a strongly voltage-dependent manner. The average hypoxic inhibition of the calcium current in 30% +/- 10% at -20 mV but only 2% +/- 2% at +30 mV. The differential inhibition of K+ and Ca2+ channels by hypoxia helps to explain why the secretory response of the cells is displaced toward PO2 values (below approximately 70 mm Hg) within the range of those normally existing in arterial blood. These data provide a conceptual framework for understanding the cellular mechanisms of O2 chemotransduction in the carotid body.     

Inhibition of Calcium Uptake, Sodium Uptake, and Catecholamine Secretion by Methoxyverapamil (D600) in Primary Cultures of Adrenal Medulla Cells

The calcium-entry antagonist D600 (methoxyverapamil) inhibited nicotine- and veratridine-induced 45Ca2+ uptake, 22Na+ uptake, and catecholamine secretion in primary cultures of bovine adrenal medulla cells. Inhibition of nicotine-induced effects occurred at D600 concentrations approximately 3-10-fold lower than those needed to produce similar inhibition of veratridine-induced effects. Inhibition of the veratridine-induced effects was competitive, but inhibition of the nicotine-induced effects was not competitive. These results suggest that D600, in addition to blocking "slow" Ca2+ channels and tetrodotoxin-sensitive Na+ channels also blocks nicotine transmission, possibly either by noncompetitively inhibiting the interaction of nicotine with the receptor binding site or by blockade of the receptor-associated ion conductance channel.     

Endoplasmic reticulum calcium signaling in nerve cells

The endoplasmic reticulum (ER) is an important organelle involved in various types of signaling in nerve cells. The ER serves as a dynamic Ca2+ pool being thus involved in rapid signaling events associated with cell stimulation by either electrical (action potential) or chemical (neurotransmitters) signals. This function is supported by Ca2+ release channels (InsP3 and ryanodine receptors) and SERCA Ca2+ pumps residing in the endomembrane. In addition the ER provides a specific environment for the posttranslational protein processing and transport of various molecules towards their final destination. In parallel, the ER acts as a "calcium tunnel," which facilitates Ca2+ movements within the cell by avoiding cytoplasmic routes. Finally the ER appears as a source of numerous signals aimed at the nucleus and involved in long-lasting adaptive cellular responses. All these important functions are controlled by intra-ER free Ca2+ which integrates various signaling events and establishes a link between fast signaling, associated with ER Ca2+ release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. Disruption of ER Ca2+ homeostasis triggers several forms of cellular stress response and is intimately involved in neurodegeneration and neuronal cell death.     

Rapid modulation of Ca2+ uptake in human jejunal enterocytes

The active metabolite of D vitamin, 1,25(OH)2D3, has been suggested to promote acute uptake of calcium through the intestinal lining in cell lines and murine models. In this study, the effects of D vitamin on the cytoplasmic Ca2+ of single human jejunal enterocytes, obtained with LOC-I-GUT technique, was analyzed in vivo in a fluorometric system using fura-2 as the Ca2+-sensing probe. Vitamin-promoted acute Ca2+ influx exhibited dual kinetics, indicating initial release from intracellular Ca2+ pools and fast entry from the extracellular space. Furthermore, providing a chemical clamp of membrane potential close to 0 mV did not activate voltage-sensitive calcium channels in the cellular membrane, neither was the hormone-induced Ca2+ influx affected by verapamil. This advocates that voltage-operated channels like L-type Ca2+ channels do not participate in the process of Ca2+ uptake. In fact, the existence of calcium-release-activated-calcium channels (I(CRAC)) was implied by the findings that irreversible depletion of intracellular Ca2+ stores by thapsigargin promoted Ca2+ entry. In the thapsigargin-treated enterocytes, D vitamin lost its ability to promote calcium entry indicating an important role for intracellular store-operated Ca2+ stores in the acute effects of 1,25(OH)2D3.     

Effects of energy deprivation and hydrogen peroxide on contraction and myoplasmic free calcium concentrations in isolated myocardial muscle cells

The effect of energy deprivation and H2O2 on the contraction, shape, and intracellular free Ca2+ concentration of myocardial muscle cells was investigated using suspensions of freshly isolated, electrically stimulated rat ventricle heart cells. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to decrease the rate of ATP synthesis. At 0.9 mM extracellular Ca2+, CCCP (0.25 microM) reduced the number of contracting cells by 50% after 5 min, and the number of rod-shaped cells by 40% after 10 min. The effects of CCCP were associated with a substantial decrease in measured cellular ATP concentrations. The deleterious effect of exposure of myocytes to CCCP for periods of up to 5 min was enhanced by an increase in the extracellular Ca2+ concentration, but markedly reduced in the absence of electrical stimulation. Verapamil protected myocytes from the deleterious effects of CCCP during the first 5 min but not at later times. In the presence of 46 mM extracellular K+, CCCP caused a marked increase in the myoplasmic free Ca2+ concentration (measured using quin2). This effect was inhibited by verapamil and was not observed in the absence of K+-induced depolarization. Exposure of myocytes to H2O2 (0.5 mM) caused a substantial decrease both in the number of cells which exhibited normal end-to-end synchronous contraction and in the total number of cells which contracted either partially or fully. The effects of H2O2 were more pronounced at higher concentrations of the peroxide, with longer times of exposure to the agent, and at higher concentrations of extracellular Ca2+, and were partially reversed by dimethyl sulfoxide. The results indicate that both ATP deprivation and H2O2, possibly through the generation of free radicals, cause substantial and rapid damage to cardiac myocytes and induce the movement of additional Ca2+ across the sarcolemma to the myoplasm. In the case of ATP deprivation, this initially occurs through voltage-operated channels.     

Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.