Role Of Cytosolic Ca2 Research Articles

Regulation of neuronal energy metabolism by calcium: Role of MCU and Aralar/malate-aspartate shuttle

Calcium is a major regulator of cellular metabolism. Calcium controls mitochondrial respiration, and calcium signaling is used to meet cellular energetic demands through energy production in the organelle. Although it has been widely assumed that Ca2+-actions require its uptake by mitochondrial calcium uniporter (MCU), alternative pathways modulated by cytosolic Ca2+ have been recently proposed. Recent findings have indicated a role for cytosolic Ca2+ signals acting on mitochondrial NADH shuttles in the control of cellular metabolism in neurons using glucose as fuel. It has been demonstrated that AGC1/Aralar, the component of the malate/aspartate shuttle (MAS) regulated by cytosolic Ca2+, participates in the maintenance of basal respiration exerted through Ca2+-fluxes between ER and mitochondria, whereas mitochondrial Ca2+-uptake by MCU does not contribute. Aralar/MAS pathway, activated by small cytosolic Ca2+ signals, provides in fact substrates, redox equivalents and pyruvate, fueling respiration. Upon activation and increases in workload, neurons upregulate OxPhos, cytosolic pyruvate production and glycolysis, together with glucose uptake, in a Ca2+-dependent way, and part of this upregulation is via Ca2+ signaling. Both MCU and Aralar/MAS contribute to OxPhos upregulation, Aralar/MAS playing a major role, especially at small and submaximal workloads. Ca2+ activation of Aralar/MAS, by increasing cytosolic NAD+/NADH provides Ca2+-dependent increases in glycolysis and cytosolic pyruvate production priming respiration as a feed-forward mechanism in response to workload. Thus, except for glucose uptake, these processes are dependent on Aralar/MAS, whereas MCU is the relevant target for Ca2+ signaling when MAS is bypassed, by using pyruvate or β-hydroxybutyrate as substrates.

Mitofusin 1 and 2 differentially regulate mitochondrial function underlying Ca2+ signaling and proliferation in rat aortic smooth muscle cells

Mitochondria play a substantial role in cytosolic Ca2+ buffering and energy metabolism. We recently demonstrated that mitofusin 2 (Mfn2) regulated Ca2+ signaling by tethering mitochondria and sarcoplasmic reticulum (SR), and thus, facilitated mitochondrial function and the proliferation of vascular smooth muscle cells (VSMCs). However, the physiological role of mitofusin 1 (Mfn1) on Ca2+ signaling and mitochondrial function remains unclear. Herein, the roles of Mfn1 and Mfn2 in mitochondrial function underlying Ca2+ signaling, ATP production, and cell proliferation were examined in rat aortic smooth muscle A10 cells. Following an arginine vasopressin-induced increase in cytosolic Ca2+ concentration ([Ca2+]cyt), Mfn2 siRNA (siMfn2) reduced cytosolic Ca2+ removal and mitochondrial Ca2+ uptake. However, Mfn1 siRNA (siMfn1) attenuated mitochondrial Ca2+ uptake, facilitated Ca2+ removal from mitochondria, and resulted in increased [Ca2+]cyt, which was mediated by the downregulation of mitochondrial Ca2+ uniporter (MCU) expression and the upregulation of mitochondrial Na+/Ca2+ exchanger (NCLX) expression. Furthermore, siMfn1 increased the mitochondrial membrane potential, ATP production by adenine nucleotide translocase (ANT), and cell proliferation, whereas siMfn2 exhibited the opposite responses. In conclusion, Mfn1 modulates the expressions of MCU, NCLX, and ANT, and Mfn2 tethers mitochondria to SR, which demonstrates their different mitochondrial functions for Ca2+ signaling, ATP production, and the proliferation of VSMCs.

Mitochondria modulate ameloblast Ca2+ signaling.

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation-stage ameloblasts focusing on their role in Ca2+ signaling. Quantification of the Ca2+ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca2+ pools by adenosine triphosphate in rhod2AM-loaded cells showed similar mitochondrial Ca2+ (m Ca2+ ) uptake. However, m Ca2+ extrusion via Na+ -Li+ -Ca2+ exchanger was more prominent in maturation. To address if m Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) played a role in cytosolic Ca2+ (c Ca2+ ) buffering, we stimulated Ca2+ influx via the store-operated Ca2+ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented c Ca2+ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher m Ca2+ uptake in maturation ameloblasts. The uptake of Ca2+ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca2+ handling in maturation but only upon stimulation of Ca2+ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca2+ physiology, but also advance the concept that SOCE and m Ca2+ uptake are complementary processes in biological mineralization.

A voltage-dependent Ca2+ homeostat operates in the plant vacuolar membrane.

Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca2+ signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca2+ and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca2+ concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H+ /Ca2+ exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca2+ homeostat could contribute to calcium signalling when coupled to a recently discovered K+ channel-dependent module for electrical excitability of the vacuolar membrane.

Targeting salt stress coping mechanisms for stress tolerance in Brassica: A research perspective

Brassica genus comprises numerous cultivated brassica species with various economic importance. Salt stress is an overwhelming problem causing serious losses in Brassica species (e.g. B. napus, B. rapa, B. oleracea, B. juncea) growth and grain yield production by inducing ionic and ROS toxicity. Given that a significant variation exists in salt tolerance level in Brassica genus, Brassica species exhibited numerous salt tolerance mechanisms which were either overlooked or given less importance to improve and understand innate salt stress tolerance mechanism in Brassica species. In this review, we tried to highlight the importance and recent findings relating to some overlooked and potential mechanisms such as role of neurotransmitters, and role of cytosolic Ca2+ and ROS as signaling elements to enhance salt stress tolerance. Studies revealed that salt tolerant brassica species retained more K+ in leaf mesophyll which confers overall salinity tolerance in salt tolerance brassica species. Neurotransmitter such as melatonin, dopamiane and eATP regulates K+ and Ca2+ permeable ion channels and plays a very crucial role in ionic homeostasis under salinity stress in brassica. At the end, the numerous possible salt stress agronomic strategies were also discussed to mitigate the severity of the salt stress in Brassica species.

Model-driven discovery of calcium-related protein-phosphatase inhibition in plant guard cell signaling.

The plant hormone abscisic acid (ABA) promotes stomatal closure via multifarious cellular signaling cascades. Our previous comprehensive reconstruction of the stomatal closure network resulted in an 81-node network with 153 edges. Discrete dynamic modeling utilizing this network reproduced over 75% of experimental observations but a few experimentally supported results were not recapitulated. Here we identify predictions that improve the agreement between model and experiment. We performed dynamics-preserving network reduction, resulting in a condensed 49 node and 113 edge stomatal closure network that preserved all dynamics-determining network motifs and reproduced the predictions of the original model. We then utilized the reduced network to explore cases in which experimental activation of internal nodes in the absence of ABA elicited stomatal closure in wet bench experiments, but not in our in silico model. Our simulations revealed that addition of a single edge, which allows indirect inhibition of any one of three PP2C protein phosphatases (ABI2, PP2CA, HAB1) by cytosolic Ca2+ elevation, resolves the majority of the discrepancies. Consistent with this hypothesis, we experimentally show that Ca2+ application to cellular lysates at physiological concentrations inhibits PP2C activity. The model augmented with this new edge provides new insights into the role of cytosolic Ca2+ oscillations in stomatal closure, revealing a mutual reinforcement between repeated increases in cytosolic Ca2+ concentration and a self-sustaining feedback circuit inside the signaling network. These results illustrate how iteration between model and experiment can improve predictions of highly complex cellular dynamics.

Anti-Cancer Effects of 3, 3'-Diindolylmethane on Human Hepatocellular Carcinoma Cells Is Enhanced by Calcium Ionophore: The Role of Cytosolic Ca2+ and p38 MAPK.

Purpose: 3,3′-Diindolylmethane (DIM), derived from indole-3-carbinol (I3C) in the Brassica species of cruciferous vegetables, has anticancer effects, but its exact underlying mechanism of action is unknown. We explored the roles of cytosolic free calcium ([Ca2+]i) and p38 MAPK in the anti-cancer effects of DIM in human hepatocellular carcinoma cells. Methods: Cell proliferation was measured with a Cell Counting Kit-8 (CCK-8) and the clonogenic formation assay. Cell apoptosis was examined by flow cytometric analysis and Hoechst dye staining. Cleaved-caspase3, cleaved-PARP, Bax, total, and phosphorylated p38 MAPK were assayed by western blotting. [Ca2+]i was measured with Fluo-3/AM by fluorescence microscopy. A23187, a calcium ionophore, was used to increase [Ca2+]i levels. Results: DIM inhibited cell proliferation in both SMMC-7721 and HepG2 cells in a concentration- and time-dependent manner. DIM also enhanced phosphorylation of p38 MAPK (p-p38), which was attenuated by SB203580. The proliferation inhibition and apoptosis induction by DIM were also blunted. In addition, DIM increased [Ca2+]i in HCC cells, and this effect was inhibited by the calcium chelator, BAPTA-AM, resulting in reduced p-p38 MAPK activation and apoptosis in DIM-treated cells, though the proliferation inhibition by DIM was unchanged. However, the DIM-induced cell proliferation inhibition and apoptosis were significantly enhanced by A23187, a selective calcium ionophore, which was attributed to exaggerated p-p38 MAPK. Conclusions: The calcium ionophore enhanced DIM-induced anti-cancer effects in hepatocellular carcinoma cells, secondary to [Ca2+]i-dependent activation of p38 MAPK. Treatment with a combination of DIM and calcium ionophore may offer a new approach to enhance the chemotherapeutic efficacy in liver cancer.

Differences in mitochondrial Ca2+ handling during challenges of CaCl2 pulses in brain synaptic and non‐synaptic mitochondria: implications for differential Ca2+ buffering

Mitochondria play a key role in cytosolic Ca2+ regulation and buffering, with Ca2+ uptake mainly via the inner membrane mitochondrial Ca2+ uniporter (MCU), and efflux of mitochondrial Ca2+ largely via the Na+/Ca2+ exchanger (mNCE). Previous reports have shown brain synaptic and non‐synaptic mitochondria display marked differences in Ca2+ uptake before permeabilization of the mitochondrial membranes, the so‐called permeable transition pore (mPTP). However, the kinetics of mitochondrial Ca2+ handling and its implications for Ca2+ buffering after boluses of CaCl2 have not been reported. In this study, we aimed to define the kinetics of Ca2+ handling in synaptic and non‐synaptic mitochondria isolated from rat brains via discontinuous percoll centrifugation followed by differential centrifugation. Respiratory control index (RCI), and subsequent changes in calcium retention capacity (CRC) and membrane potential (ΔΨm), were assessed in isolated mitochondria. CRC and ΔΨm were evaluated with Fura 4F penta‐K salt and TMRM dyes, respectively, using fluorescence spectrophotometry (Photon Technology). Mitochondria were energized with glutamate and malate, and CRC and ΔΨm were assessed during state 2 respiration. To investigate the potential role of the mNCE in Ca2+ handling, the NCE blocker CGP37157 (CGP) was used. In addition, experiments were conducted to determine the expression levels of the MCU and mNCE in the two mitochondria pools using western blot. Mitochondrial RCI was higher in the synaptic group compared to the non‐synaptic group, which is consistent with past results. Ca2+ uptake and retention without CGP were lower in synaptic mitochondria compared to non‐synaptic mitochondria. Addition of CGP markedly enhanced CRC in the synaptic mitochondria to levels found in non‐synaptic mitochondria without CGP. In contrast, the CGP induced changes in CRC were not observed in the non‐synaptic mitochondria. Membrane potential depolarization occurred in both synaptic and non‐synaptic mitochondria as matrix free Ca2+ accumulated, reflecting the decrease in Ca2+ sequestration or Ca2+ efflux in the non‐synaptic and synaptic mitochondria, respectively. MCU expression was higher in the non‐synaptic mitochondria compared to the synaptic mitochondria, whereas mNCE expression was not different. The increased Ca2+ uptake and retention in the presence of CGP in the synaptic mitochondria implies that mNCE is active in synaptic mitochondria, which leads to faster extrusion of added Ca2+ before it is sequestered. The lack of change in Ca2+ kinetics in non‐synaptic mitochondria suggests that NCE activity is lower. The ΔΨm and CRC results likely indicate that synaptic mitochondria can buffer Ca2+ like that in non‐synaptic mitochondria, but only do so when the mNCE is blocked. We propose that synaptic mitochondria may have increased mNCE activity to regulate its matrix free Ca2+, while non‐synaptic mitochondria depend mostly on buffering of the free Ca2+; the implications for these differential responses may be related to the physiological role of these two populations of mitochondria.Support or Funding InformationFunding: NIH T35 HL072483 and MCW‐AHWThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Luminal Ca2+ dynamics during IP3R mediated signals

The role of cytosolic Ca2+ on the kinetics of Inositol 1,4,5-triphosphate receptors (IP3Rs) and on the dynamics of IP3R-mediated Ca2+ signals has been studied at large both experimentally and by modeling. The role of luminal Ca2+ has not been investigated with that much detail although it has been found that it is relevant for signal termination in the case of Ca2+ release through ryanodine receptors. In this work we present the results of observing the dynamics of luminal and cytosolic Ca2+ simultaneously in Xenopus laevis oocytes. Combining observations and modeling we conclude that there is a rapid mechanism that guarantees the availability of free Ca2+ in the lumen even when a relatively large Ca2+ release is evoked. Comparing the dynamics of cytosolic and luminal Ca2+ during a release, we estimate that they are consistent with a 80% of luminal Ca2+ being buffered. The rapid availability of free luminal Ca2+ correlates with the observation that the lumen occupies a considerable volume in several regions across the images.

Multiple cytosolic calcium buffers in posterior pituitary nerve terminals.

Cytosolic Ca(2+) buffers bind to a large fraction of Ca(2+) as it enters a cell, shaping Ca(2+) signals both spatially and temporally. In this way, cytosolic Ca(2+) buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca(2+) entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca(2+) buffers in controlling pituitary Ca(2+) signaling is poorly understood. Here, cytosolic Ca(2+) buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca(2+) indicator fluo-8 revealed stepwise increases in free Ca(2+) after a series of brief depolarizing pulses in rapid succession. These Ca(2+) increments grew larger as free Ca(2+) rose to saturate the cytosolic buffers and reduce the availability of Ca(2+) binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a Kd of 1.5-4.7 µM, and approximately half contained an additional higher-affinity buffer with a Kd of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca(2+) buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca(2+) signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones.

Identification of Novel Ryanodine Receptor 1 (RyR1) Protein Interaction with Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP)

The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca(2+) release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1-4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca(2+) release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling.

Cholesterol efflux to apoA-I in ABCA1-expressing cells is regulated by Ca2+-dependent calcineurin signaling

ATP-binding cassette transporter A1 (ABCA1) is required for the lipidation of apolipoprotein A-I (apoA-I), although molecular mechanisms supporting this process remain poorly defined. In this study, we focused on the role of cytosolic Ca(2+) and its signaling and found that cytosolic Ca(2+) was required for cholesterol efflux to apoA-I. Removing extracellular Ca(2+) or chelating cytosolic Ca(2+) were equally inhibitory for apoA-I lipidation. We provide evidence that apoA-I induced Ca(2+) influx from the medium. We further demonstrate that calcineurin activity, the downstream target of Ca(2+) influx, was essential; inhibition of calcineurin activity by cyclosporine A or FK506 completely abolished apoA-I lipidation. Furthermore, calcineurin inhibition abolished apoA-I binding and diminished JAK2 phosphorylation, an established signaling event for cholesterol efflux to apoA-I. Finally, we demonstrate that neither Ca(2+) manipulation nor calcineurin inhibition influenced ABCA1's capacity to release microparticles or to remodel the plasma membrane. We conclude that this Ca(2+)-dependent calcineurin/JAK2 pathway is specifically responsible for apoA-I lipidation without directly modifying ABCA1 activity.

Downstream from calcium signalling: mitochondria, vacuoles and pancreatic acinar cell damage

Ca(2+) is one of the most ancient and ubiquitous second messengers. Highly polarized pancreatic acinar cells serve as an important cellular model for studies of Ca(2+) signalling and homeostasis. Downstream effects of Ca(2+) signalling have been and continue to be an important research avenue. The primary functions regulated by Ca(2+) in pancreatic acinar cells--exocytotic secretion and fluid secretion--have been defined and extensively characterized in the second part of the last century. The role of cytosolic Ca(2+) in cellular pathology and the related question of the interplay between Ca(2+) signalling and bioenergetics are important current research lines in our and other laboratories. Recent findings in these interwoven research areas are discussed in the current review.

Destabilization of parathyroid hormone mRNA by extracellular Ca2+ and the calcimimetic R-568 in parathyroid cells: role of cytosolic Ca and requirement for gene transcription

Extracellular Ca reduces parathyroid hormone (PTH) levels through several mechanisms, but many details of the intracellular steps involved have been difficult to elucidate because of the lack of a suitable parathyroid cell model. The present studies utilized our Ca-responsive bovine parathyroid organoid culture system (pseudoglands) to examine PTH mRNA in intact parathyroid cells. Increasing medium calcium from 0.4 to 3.0 mM reduced PTH mRNA to 20-30% of basal by 16 h. Reducing medium Ca from 3.0 to 0.4 mM restored PTH mRNA levels over a 24-h period. PTH mRNA was also reduced by the calcimimetic R-568, confirming the role of the calcium-sensing receptor. PTH decay rates were determined by placing pseudoglands in either 0.4 or 3.0 mM Ca for 2 h and then blocking gene transcription. PTH mRNA remained stable for at least 24 h in pseudoglands incubated in 0.4 mM Ca, but fell gradually by 62% in the presence of 3.0 mM Ca. Blocking transcription prior to the addition of high-Ca medium dramatically blunted the Ca-induced degradation of PTH mRNA, indicating that acceleration of PTH mRNA decay by Ca requires gene transcription. Pharmacologic investigation of the signaling pathways involved indicated that the Ca-induced reduction of PTH mRNA did not involve MAP kinase, phospholipase D, or cyclic AMP. However, increasing cytosolic Ca with thapsigargin or the Ca ionophore A23187 decreased PTH mRNA levels. In summary, Ca-mediated destabilization of PTH mRNA requires gene transcription and involves increases in cytosolic Ca.

Developing Sensors for Real-Time Measurement of High Ca2+ Concentrations

Ca2+ regulates numerous biological processes through spatiotemporal changes in the cytosolic Ca2+ concentration and subsequent interactions with Ca2+ binding proteins. The endoplasmic reticulum (ER) serves as an intracellular Ca2+ store and plays an essential role in cytosolic Ca2+ homeostasis. There is a strong need to develop Ca2+ sensors capable of real-time quantitative Ca2+ concentration measurements in specific subcellular environments without using natural Ca2+ binding proteins such as calmodulin, which themselves participate as signaling molecules in cells. In this report, a strategy for creating such sensors by grafting a Ca2+-binding motif into chromophore sensitive locations in green fluorescence protein is described. The engineered Ca2+ sensors exhibit large ratiometric fluorescence and absorbance changes upon Ca2+ binding with affinities corresponding to the Ca2+ concentrations found in the ER (Kd values range from 0.4 to 2 mM). In addition to characterizing the optical and metal binding properties of the newly developed Ca2+ sensors with various spectroscopic methods, we also examined the kinetic properties using stopped-flow spectrofluorimetry to ensure accurate monitoring of dynamic Ca2+ changes. The developed Ca2+ sensor was successfully targeted to the ER of mammalian cell lines to monitor Ca2+ changes occurring in this compartment in response to stimulation with agonists. We envision that this class of Ca2+ sensors can be modified further to measure the Ca2+ concentration in other cellular compartments, providing tools for studying the contribution of these compartments to cellular Ca2+ signaling.

Stretch‐induced TRPC4 downregulation in RASM cells may be due to changes in intracellular calcium

Our previous work showed that cyclic stretch significantly decreases TRPC4 expression in rat aortic smooth muscle (RASM) cells and is prevented by CPA. TRPC4 downregulation may be a compensatory mechanism to reduce increased [Ca2+]SR resulting from entry through stretch-activated channels. However, we do not yet know the role of cytosolic Ca2+. We hypothesized that TRPC4 downregulation may also serve to reduce stretch-induced increases in cytosolic [Ca2+] and outlined the following aims: (1) demonstrate that increased cytosolic [Ca2+] in the absence of stretch can decrease TRPC4 protein expression, and (2) establish whether blocking Ca2+ entry into the cytosol can prevent stretch-induced TRPC4 downregulation. RASM cells were subjected to cyclic stretch for 6h using the Flexercell Strain Unit FX-4000. Control cells were not stretched. To address Aim (1), the [Ca2+] of the cell culture medium was increased to 10 mM in both stretch and non-stretch conditions, and TRPC4 expression was measured by Western blot. 10 mM Ca2+ decreased TRPC4 protein expression in the absence of stretch (p<0.05). However, high Ca2+ conditions in the presence of stretch did not show any additional downregulation. To address Aim (2), gadolinium was added before mechanical strain to block stretch-activated channels. Nifedipine was used to assess the contribution of L-type channels. Addition of either drug in the absence of stretch increased TRPC4 expression. However, stretch-induced downregulation was still present within each drug condition. These results suggest that TRPC4 proteins may play an important role in stabilizing Ca2+ levels in RASM cells during mechanical or pharmacological changes. Support: COBRE P20 RR18766 “Mentoring in Cardiovascular Biology”

Effect of action potential on photosynthesis and spatially distributed H+ fluxes in cells and Chloroplasts of Chara corallina

When exposed to light, the cells of characean algae produce intermittent regions of H+ extrusion and H+ absorption, featuring different photosynthetic activities. Methods for local measurements of outer pH, O2 content, and photochemical activity of photosystem II (PSII) were applied to examine microscopic regions of Chara coralline Klein ex Willd. internodes. The results show that the functional spatial heterogeneity of these excitable cells is controlled not only by light but also by electric excitation of the plasma membrane. Generation of a single action potential (AP) induced a reversible transition to the state with homogenous pH distribution and had different effects on photosynthesis in cell regions producing alkaline and acid zones. The effective quantum yield of PSII primary processes and the maximal chlorophyll fluorescence decreased after AP in the alkaline cell regions but were almost unaffected in the acidic cell regions. The suppression of photosynthesis after AP was also evident in the decrease of photosynthetic O2 evolution. The results provide evidence that electric signals arising at the plasmalemma are transmitted to the level of thylakoid membranes. The effects of electric excitation on fluorescence and the quantum yield of PSII photochemistry were best pronounced at low light intensities and low level of nonphotochemical quenching. The sensitivity of chlorophyll fluorescence in resting and excited cells to light intensity and protonophores indicates that the AP-induced fluorescence changes derive from the increase in pH gradient at the thylakoid membrane. The temporal elimination of alkaline zones and inhibition of photosynthesis apparently arise from parallel operational sequences that have a common initial stage. A possible role of cytosolic Ca2+ rise in the mechanism of photosynthesis suppression after electric excitation of the plasma membrane is discussed.

Ankyrin-B Coordinates the Na/K ATPase, Na/Ca Exchanger, and InsP3 Receptor in a Cardiac T-Tubule/SR Microdomain

We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor “dyads.” E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B+/− cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.

Dominant Role of Mitochondria in Calcium Homeostasis of Single Rat Pituitary Corticotropes

The rise in cytosolic free Ca2+ concentration ([Ca2+]i) is the major trigger for secretion of ACTH from pituitary corticotropes. To better understand the shaping of the Ca2+ signal in corticotropes, we investigated the mechanisms regulating the depolarization-triggered Ca2+ signal using patch-clamp techniques and indo-1 fluorometry. The rate of cytosolic Ca2+ clearance was unaffected by inhibitors of Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase (PMCA), slightly slowed by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, but dramatically slowed by mitochondrial uncouplers or inhibitor of mitochondrial uniporter. Measurements with rhod-2 revealed that depolarization-triggered increase in mitochondrial Ca2+ concentration. Thus, mitochondria have a dominant role in cytosolic Ca2+ clearance. Using the Mn2+ quench technique, we found the presence of a continuous basal Ca2+ influx in corticotropes. This basal Ca2+ influx was balanced by the combined actions of mitochondrial uniporter and PMCA and SERCA pumps. Inhibition of the mitochondrial uniporter or PMCA or SERCA pumps elevated basal [Ca2+]i. Using membrane capacitance measurement, we found that the change in the shape of the depolarization-triggered Ca2+ signal after mitochondrial inhibition was associated with enhancement of the exocytotic response. Thus, mitochondria have a dominant role in the regulation of Ca2+ signal and exocytosis in corticotropes.

Menadione-induced apoptosis: roles of cytosolic Ca(2+) elevations and the mitochondrial permeability transition pore.

In normal pancreatic acinar cells, the oxidant menadione evokes repetitive cytosolic Ca(2+) spikes, partial mitochondrial depolarisation, cytochrome c release and apoptosis. The physiological agonists acetylcholine and cholecystokinin also evoke cytosolic Ca(2+) spikes but do not depolarise mitochondria and fail to induce apoptosis. Ca(2+) spikes induced by low agonist concentrations are confined to the apical secretory pole of the cell by the buffering action of perigranular mitochondria. Menadione prevents mitochondrial Ca(2+) uptake, which permits rapid spread of Ca(2+) throughout the cell. Menadione-induced mitochondrial depolarisation is due to induction of the permeability transition pore. Blockade of the permeability transition pore with bongkrekic acid prevents activation of caspase 9 and 3. In contrast, the combination of antimycin A and acetylcholine does not cause apoptosis but elicits a global cytosolic Ca(2+) rise and mitochondrial depolarisation without induction of the permeability transition pore. Increasing the cytosolic Ca(2+) buffering power by BAPTA prevents cytosolic Ca(2+) spiking, blocks the menadione-elicited mitochondrial depolarisation and blocks menadione-induced apoptosis. These results suggest a twin-track model in which both intracellular release of Ca(2+) and induction of the permeability transition pore are required for initiation of apoptosis.