SERCA Pump Research Articles

Calcium Alternans in a Couplon Network Model of Ventricular Myocytes: Roles of Sarcoplasmic Reticulum Load

Intracellular calcium (Ca) alternans of cardiac myocytes has been shown in many experimental studies, and the mechanisms remain incompletely understood. We recently developed a “3R theory” in which alternans arises as a result of the interactions of three critical properties: Randomness of Ca sparks; Recruitment of a Ca spark to its neighboring Ca release units (CRUs); and Refractoriness of the CRU. On the other hand, experimental studies have shown that sarcoplasmic reticulum (SR) Ca load plays important roles in the genesis of Ca alternans. In this study, we use computer simulation to study how SR Ca load and other physiological parameters, such as RyR sensitivity, SERCA pump, CRU spacing, Na-Ca exchange strength, L-type Ca conductance, etc., affect Ca alternans. Our model consists of 100 x 20 x 10 CRUs coupled via Ca diffusion in the cytoplasmic and SR space. Each CRU contains 100 RyRs with individual RyRs simulated stochastically. We developed a method to calculate the primary spark rate and the recruitment rate, and paced the model to periodically to elicit Ca alternans. We show that altering the physiological parameters not only directly change the 3 R's but also alters the SR Ca load (or the total Ca of the cell), and thus Ca alternans properties. However, higher SR Ca load causes more Ca leak which in turn causes RyRs to be more sensitive to cytosolic Ca, affecting primary spark rate and recruitment. Therefore, our present study shows that although the 3R theory does not include the SR load per se, the SR Ca load affects Ca alternans via its effects on the 3 R's.

Mathematical Model of Oxygen Labeling to Study Heart Energy Transfer

The mechanisms underlying the homeostasis of ATP, ADP, PCr and inorganic phosphate in heart under a wide range of workloads remain unclear. Fundamental to this search is an accurate understanding of the recycling fluxes of these metabolites between the mitochondrial inner membrane space and the ATPases on both the myofibrils and SERCA pumps. In addition to 31P-NMR inversion and saturation transfer studies, dynamic 18O labeling data has been used to analyze energy transfer. An integrative kinetic model that tracks the mass isotopomers of these four metabolites was constructed with the following compartments: mitochondrial matrix, mitochondrial intermembrane space, cytosol, and enzyme bound states of both ATPase and ATP synthase (since these reactions are not unidirectional with respect to oxygen exchange). A sensitivity analysis of this system was conducted with a jump to 30% H218O to find flux parameters that influenced the 18O labeling state. Both the creatine kinase and adenylate kinase shuttle fluxes were modeled as being bidirectional to test assumptions made in previous studies that analyzed dynamic 18O labeling data. It was found that the sum of forward and reverse fluxes through each shuttle determined the labeling state such that if the sum is kept constant and the net flux is reduced, a very similar labeling state is predicted. Total creatine kinase and adenylate kinase fluxes that exceeded the ATP synthase rate were also found to give almost the same predictions of the labeling state. Model predictions of the 18O labeling state of metabolites are very similar using energy fluxes reported in earlier studies of 18O labeling in the heart and energy transfer analysis by 31P-NMR inversion and saturation transfer.

Feed Forward Modeling. Fixing the Force Frequency Relationship

The force frequency relationship has intrigued researchers since its discovery by Bowditch in 1871. Several attempts were made to construct mathematical descriptions of this phenomenon; from the simple formulation of Koch-Wesser & Blinks in 1963 to the most sophisticated ones of present days. This property of the cardiac muscle is amplified by the β adrenergic stimulation. In a coordinated way the neurohumoral state alters both: frequency (acting on the SA node) and force generation (modifying the ventricular myocytes). This synchronized tuning is needed to meet new metabolic demands. Failure to do so has deleterious consequences. We implemented this physiological coordination in a new version of LabHEART (v5.5) where the cell parameters are updated according to the frequency of stimulation in a sigmoidal fashion (namely: ICaL, IKs, SERCA pump and myofilaments’ Ca-sensitivity). This feed forward modeling helps to reproduce a more realistic cell behavior and complement the information obtained by experiments where frequency has been altered but without adding β-adrenergic agonists. Plots like rate-adaptation or APD vs cycle length can, now, be mapped into the neurohumoral state to provide insights about arrhythmias (or antiarrhythmic drugs). Cardiac modelers have already linked the mechanical and the electrical activity in their formulations and showed how those activities feedback on each other. Feed forward modeling is a step towards reproducing the neurological control and thus, to have a complete description of the heart performance, particularly when changes in frequency are involved. The presumptive sigmoidal dependence of parameters on pacing frequency used here can be refined but is a valid starting point to implement the convergence of cardiac chronotrophy and inotropy.

Transitions Among Excitable, Oscillatory and Non-Excitable in Simplified Calcium Cycling Model

Calcium (Ca) plays a central role in cardiac excitation-contraction coupling. Under normal conditions, Ca entering through the L-type Ca channel opens ryanodine receptors (RyRs), which are regulated by cytosolic ([Ca]i) and luminal Ca ([Ca]SR), and causes Ca release from the sarcoplasmic reticulum (SR). This positive feedback by Ca induced Ca release makes the Ca cycling excitable. However, as the cell goes to pathological conditions, Ca releases from the SR occurs spontaneously, leading to Ca oscillation. This transition from excitable to oscillatory can be caused by different factors, including Ca overload, RyR sensitization, SERCA uptake regulation, and structural remodeling etc. Here we use a simplified model of intracellular Ca cycling to investigate how each factor contributes to Ca cycling dynamics and shifts the system among excitable, oscillatory and non-excitable states. We find that: Increase of RyR sensitivity decreases the [Ca]SR threshold for Ca oscillation. When [Ca]SR is far from the threshold, increasing RyR sensitivity only is not enough to induce oscillation. As the system approaches [Ca]SR threshold, increasing RyR sensitivity only can induce oscillation by changing the system from excitable to oscillatory. In the oscillatory regime, increasing RyR sensitivity leads to more frequent oscillation at low [Ca]SR; However, if the RyR sensitivity becomes too low or too high, Ca oscillation is abolished. 2) Below the threshold, increase in extracellular [Ca] ([Ca]o) leads to increase in [Ca]SR and release amplitude; Above the [Ca]SR threshold, increase in [Ca]o leads to increase in oscillation frequency, decreases in oscillation amplitude and basal [Ca]SR. 3) Increase of SERCA pump increases the threshold for Ca oscillation; If SERCA is too low it cannot support Ca oscillation.

Coupling of P2Y Mediated Store Depletion and Ca2+ Entry (SOCE) to KCa3.1 (KCnn4/SK4/Ik) Channel Activation in Microglia

We recently published that SK3/KCa2.3 and SK4/KCa3.1 channels contribute to microglial activation and neurotoxicity via regulation of p38 MAPK activation; and were intrigued that two SK channels are involved in the same outcomes. Cloned SK channels are activated by similar rises in Ca2+; thus, we asked whether they are functionally coupled to different Ca2+ sources. Damaged neurons release ATP, a purinergic receptor ligand that activates microglia by raising intracellular Ca2+. Purinergic responses are mediated by ionotropic P2X or metabotropic P2Y receptors. Here, we stimulated MLS-9 cells (a microglia cell line that expresses both SK3 and SK4) with UTP to trigger P2Y-mediated responses; i.e., depletion of intracellular Ca2+ stores, and subsequent store operated Ca2+ entry (SOCE). In Fura-2 loaded cells, UTP evoked a rapid Ca2+ transient, followed by a second, more sustained rise due to SOCE. The second phase was decreased by the inhibitor 2-APB, which we recently showed blocks Ca2+ release-activated Ca2+ (CRAC) channels in microglia. In perforated-patch recordings, UTP activated a robust current, which was blocked by the SK4 inhibitor, TRAM-34 (but not by the SK3 blocker, apamin), and was therefore, SK4/KCa3.1. SK4 activation by UTP was also inhibited by 2-APB, suggesting that SOCE is required for, and coupled to, SK4 channel activation. SK4 activity was apparently needed for Ca2+ entry and store re-filling; the SK4 blocker, TRAM-34, accelerated the decay in internal Ca2+ after UTP, and reduced the amplitude of a second Ca2+ release from stores evoked by the SERCA pump inhibitor, thapsigargin. Our data support selective coupling of SK4 channels (but not SK3) to Ca2+ entry through SOCE/CRAC. Our current model is that SK4 activation maintains a hyperpolarized membrane potential that helps drive Ca2+ entry through CRAC, facilitating store refilling.

Store operated Ca2+ entry dependent contraction of coronary artery smooth muscle: Inhibition by peroxide pretreatment

The sarco/endoplasmic reticulum (SER) Ca2+ pool is refilled by the SER Ca2+ pump (SERCA) using cytosolic Ca2+ and/or extracellular Ca2+ entering the cell. The effects of the SERCA pump inhibitor cyclopiazonic acid (CPA) were studied in pig coronary artery smooth muscle using two protocols. In protocol A, the SERCA pump was inhibited by adding CPA to cells/tissues in Ca2+-containing solution, whereas in protocol B, CPA was added to cells/tissues in Ca2+-free solution, followed by reintroduction of extracellular Ca2+. Addition of CPA increased cytosolic Ca2+ in cultured smooth muscle cells and elicited contraction in de-endothelialized coronary arteries in both protocols. Based on pharmacological experiments, the CPA-induced contraction of de-endothelialized arteries in protocol B resulted from store operated Ca2+ entry (SOCE). Reactive oxygen species such as peroxides are known to damage the SERCA pump in this tissue. Consistently, CPA-induced contractions were decreased in arteries pre-treated with hydrogen peroxide in protocol A. However, this pretreatment also decreased the force of contraction due to SOCE in protocol B, suggesting that it closed SOCE. We propose that the closure of SOCE triggered by exposure to reactive oxygen species may be a protective mechanism, so that Ca2+ entry by this pathway is disallowed when SERCA is damaged in pathologies such as ischemia–reperfusion.

Arachidonic acid mobilizes Ca2+ from the endoplasmic reticulum and an acidic store in rat pancreatic β cells

In rat pancreatic β cells, arachidonic acid (AA) triggered intracellular Ca2+ release. This effect could be mimicked by eicosatetraynoic acid, indicating that AA metabolism is not required. The AA-mediated Ca2+ signal was not affected by inhibition of ryanodine receptors or emptying of ryanodine-sensitive store but was reduced by ∼70% following the disruption of acidic stores (treatment with bafilomycin A1 or glycyl-phenylalanyl-β-naphthylamide (GPN)). The action of AA did not involve TRPM2 channels or NAADP receptors because intracellular dialysis of adenosine diphosphoribose (ADPR; an activator of TRPM2 channels) or NAADP did not affect the AA response. In contrast, stimulation of IP3 receptors via intracellular dialysis of adenophostin A, or exogenous application of ATP largely abolished the AA-mediated Ca2+ signal. Intracellular dialysis of heparin abolished the ATP-mediated Ca2+ signal but not the AA response, suggesting that the action of AA did not involve the IP3-binding site. Treatment with the SERCA pump inhibitor, thapsigargin, reduced the amplitude of the AA-mediated Ca2+ signal by ∼70%. Overall, our finding suggests that AA mobilizes Ca2+ from the endoplasmic reticulum as well as an acidic store and both stores could be depleted by IP3 receptor agonist. The possibility of secretory granules as targets of AA is discussed.

Role of activating transcription factor 3 in low glucose- and thapsigargin-induced apoptosis in cultured mouse islets

In vitro, survival and function of rat pancreatic β-cells are optimally preserved in the presence of 10mmol/l glucose (G10) and markedly altered by prolonged culture at either 2mmol/l glucose (G2) or 30mmol/l glucose (G30). The increase in islet cell apoptosis in G2 and G30 vs. G10 is preceded by parallel increases in the mRNA levels of the integrated stress response (ISR) gene activating transcription factor 3 (Atf3) and its putative target and proapoptotic gene growth arrest- and DNA damage-inducible gene 153 (Gadd153/Chop). In this study, we used islets from Atf3 knockout (Atf3−/−) mice to test the role of ATF3 in the stimulation of islet cell apoptosis under conditions associated with ISR activation. The glucose sensitivity of Atf3−/− and WT islets for the stimulation of insulin secretion and Xbp1 mRNA splicing during 18h culture was similar, demonstrating that glucose metabolism was unaffected by Atf3 deletion. However, the stimulation of islet cell apoptosis by the SERCA pump inhibitor thapsigargin was slightly but significantly reduced in Atf3−/− vs. WT islets despite similar level of expression of Gadd153 and Gadd34 mRNA. Also, the stimulation of islet cell apoptosis by 7days of culture in G2 was slightly but significantly reduced in Atf3−/− vs. WT islets, and this effect was accompanied by a significant reduction in Gadd153 mRNA expression. In conclusion, the increase in Atf3 gene expression induced by thapsigargin and low glucose concentrations slightly contributes to the stimulation of islet cell apoptosis under these culture conditions.

Contribution of SERCA and IP3 sensitivity to calcium signaling in astrocytes: a computational study

Modeling the mechanisms of astrocytic calcium signals is important, as astrocytes have an essential role in regulating the neuronal microenvironment of the central nervous system [1,2]. The results of the wet-lab and clinical studies can be complemented by mathematical models to gain better understanding of the complex molecular level interactions seen, for example, in the pathogenesis of Alzheimer’s disease (AD). In the aging brain astrocytes are known to change their phenotype [3], also their ionic equilibrium and function can be altered by the interaction of released and accumulated transmitters and peptides, such as, amyloid-β peptides [Aβ, 4]. The authors have recently shown, experimentally and computationally, that small amounts of Aβ25-35 fragment amplify the transmitter-induced calcium signals in astrocytes [5]. The reason for the amplification may be changes in calcium release from endoplasmic reticulum (ER) via, for example, changes in the function of sarco(endo)plasmic calcium adenosine 5’-triphosphatase (SERCA) pumps and/or in intracellular inositol 1,4,5-trisphosphate (IP3) sensitivity [6]. Mutations in presenilin 1 (one of the factors in familial AD involved in the accumulation of Aβ fragments in the brain) may change the activity of the SERCA, which pumps the cytosolic calcium into the ER lumen, leading eventually to higher concentration of calcium in ER [6]. Thus, the current hypothesis is that exceptional cytosolic calcium signals via ER, overfilled with calcium, may explain the calcium changes detected in the presence of Aβ. We here study the effect of SERCA pumps and IP3 sensitivity on calcium signals in astrocytes by further exploring the existing deterministic [7] and stochastic [5] models to explain the altered calcium regulation. The models include the six major mechanisms known to be involved in calcium signaling in astrocytes; 1) calcium leak from/to extracellular matrix (ECM), 2) capacitive calcium entry from ECM, 3) calcium entry via ionotropic receptors, 4) calcium leak from intracellular stores, such as ER, 5) storage of calcium to ER via SERCA pumps, and 6) calcium release from ER mediated by IP3. In this study, we computationally explore and verify the role of SERCA pump and IP3 sensitivity -induced changes in intracellular calcium signals experimentally shown in [8] and [9]. The understanding of calcium signals in astrocytes is essential as the changes in astrocytic calcium signaling are prone to cause widespread alterations in neuronal network function and can lead to neurological disorders.

New insights on sarcoplasmic reticulum calcium regulation in muscle fatigue

a consistent observation with fatigue in skeletal muscle is a decline in the amplitude of the myoplasmic Ca2+ transient, which is thought to result primarily from a reduced Ca2+ flux through the ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR) ([Fig. 1][1]). This in turn is thought to

Bradykinin acutely inhibits activity of the epithelial Na+channel in mammalian aldosterone-sensitive distal nephron

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na(+) channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability (P(o)) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg(9)-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC P(o) when G(q/11) was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of G(q/11)-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI(4,5)P(2) levels. However, inhibition of Ca(2+) pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca(2+)](i) as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC P(o) in mammalian ASDN via stimulation of B2 receptors and following depletion of PI(4,5)P(2), but not increases in [Ca(2+)](i).

PERK Activation at Low Glucose Concentration Is Mediated by SERCA Pump Inhibition and Confers Preemptive Cytoprotection to Pancreatic β-Cells

This article appears in Molecular Endocrinology, published December 30, 2010, 10.1210/me.2010-0309

PERK Activation at Low Glucose Concentration Is Mediated by SERCA Pump Inhibition and Confers Preemptive Cytoprotection to Pancreatic β-Cells

PERK Activation at Low Glucose Concentration Is Mediated by SERCA Pump Inhibition and Confers Preemptive Cytoprotection to Pancreatic β-Cells

A new Mathematical Formulation of the Plasma Membrane Ca 2+-Atpase (PMCA) Provides Stability to Simulated Ca 2+ Signaling in Human T Cells

In lymphocytes Ca2+ signals are essential for diverse cellular functions. After antigen binds to the T cell receptor a series of reactions are initiated that generate IP3 and culminate in an increase in cytosolic Ca2+. Mechanisms that remove Ca2+ also exert an important influence on the net Ca2+ level. The SERCA pump resequesters Ca2+ into the ER and the PMCA transports Ca2+ to the extracellular side. Two key features of the PMCA are its stimulation by Ca2+-calmodulin and by PKA-dependent phosphorylation (Bers, 2001) but in most of the mathematical models this pump is represented with a simple Michaelis-Menten formulation due to its small contribution to the overall Ca2+ fluxes. This is not the case in T cells, Bautista et al (2002) showed that this pump is the primary means of Ca2+ extrusion in T cells and its activity is modulated by Ca2+ enabling the cell to adapt to higher Ca2+ values during T cell activation. Additionally once [Ca2+]i returned to baseline levels the PMCA recovered slowly with a time constant of ∼ 4 min providing a “memory” of previous [Ca2+]i. We created a first order kinetics model of the PMCA that mimics its activation and recovery as function of [Ca2+]i (Vmax increased from 30 to 60 nM s−1, Km decreased from 500 to 400 nM when fully activated) and incorporated into a mathematical description of Ca2+ signaling in T cells (LymphoLAB). Our results indicate that modulation of the PMCA activity improves the stability of Ca2+ signaling by adjusting the pump rate to Ca2+ influx even at high [Ca2+]i levels (preventing a harmful Ca2+ overload). Moreover the delay in modulation permits small Ca2+ fluxes to increase transiently enhancing Ca2+ signaling dynamics.

New findings on nuclear gangliosides: overview on metabolism and function

GM1 and GD1a gangliosides occur in both membranes of the nuclear envelope (NE) together with two isoforms of neuraminidase. The Neu3 isoform of neuraminidase occurs in the inner membrane of the NE and Neu1 in the outer membrane. Both isoforms convert GD1a to GM1 within the respective membranes. GM1 in the inner membrane is tightly associated with a Na(+) /Ca(2+) exchanger (NCX) and potentiates the latter's activity. The NCX/GM1 complex mediates transfer of nucleoplasmic Ca(2+) to the NE lumen and hence to the endoplasmic reticulum (ER) with which it is continuous. Since cytoplasmic- and nucleoplasmic Ca(2+) are in homeostatic equilibrium (via nuclear pores), the nuclear NCX/GM1 complex acts to gate Ca(2+) transfer from cytosol to ER via nucleoplasm and NE. This constitutes an alternate route to the SERCA pump, indicating the influence of nuclear NCX/GM1 on whole cell Ca(2+) homeostasis. Use of cameleon-fluorescent Ca(2+) indicators (R. Tsien) demonstrated no Ca(2+) transfer from cytosol/nucleoplasm to ER in cells lacking nuclear NCX (Jurkat), and significantly reduced Ca(2+) transfer in cells lacking nuclear GM1 (NG-CR72). NCX/GM1 appears in the NE of neurons as they differentiate and serves a cytoprotective function, as seen in the high susceptibility of GalNAcT-/- knockout mice to kainate-induced seizure activity. This was alleviated by intraperitoneal injections of LIGA-20 a derivative of GM1 that is able (unlike GM1 itself) to traverse the blood brain barrier and neuronal plasma membrane and insert into the NE where it restores NCX exchanger activity. Absence or loss of nuclear GM1 renders cells vulnerable to apoptotic elimination.

PERK Activation at Low Glucose Concentration Is Mediated by SERCA Pump Inhibition and Confers Preemptive Cytoprotection to Pancreatic β-Cells

Protein kinase R-like ER kinase (PERK) is activated at physiologically low glucose concentrations in pancreatic β-cells. However, the molecular mechanisms by which PERK is activated under these conditions and its role in β-cell function are poorly understood. In this report, we investigated, in dispersed rat islets of Langerhans and mouse insulinoma-6 (MIN6) cells, the relationship between extracellular glucose concentration, the free endoplasmic reticulum (ER) calcium concentration ([Ca(2+)](ER)) measured directly using an ER targeted fluorescence resonance energy transfer-based calcium sensor, and the activation of PERK. We found that a decrease in glucose concentration leads to a concentration-dependent reduction in [Ca(2+)](ER) that parallels the activation of PERK and the phosphorylation of its substrate eukaryotic initiation factor-2α. We provide evidence that this decrease in [Ca(2+)](ER) is caused by a decrease in sarcoplasmic/ER Ca(2+)-ATPase pump activity mediated by a reduction in the energy status of the cell. Importantly, we also report that PERK-dependent eukaryotic initiation factor-2α phosphorylation at low glucose concentration plays a significant role in 1) the regulation of both proinsulin and global protein synthesis, 2) cell viability, and 3) conferring preemptive cytoprotection against ER stress. Taken together, these results provide evidence that a decrease in the ATP/energy status of the cell in response to a decrease in glucose concentration results in sarcoplasmic/ER Ca(2+)-ATPase pump inhibition, the efflux of Ca(2+) from the ER, and the activation of PERK, which plays an important role in both pancreatic β-cell function and survival.

Prion Protein Misfolding Affects Calcium Homeostasis and Sensitizes Cells to Endoplasmic Reticulum Stress

Prion-related disorders (PrDs) are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrPRES. Altered endoplasmic reticulum (ER) homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrPRES. Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrPRES and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.

Thapsigargin activates Ca 2+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

The ER Ca 2+ sensor STIM1 and the Ca 2+ channel Orai1 are key players in store-operated Ca 2+ entry (SOCE). In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926. siRNA directed against STIM1 or Orai1 reduced Ca 2+ entry by about 50–60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca 2+ entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca 2+ entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PK Ceta is required for Ca 2+ entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca 2+ entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca 2+ entry by more than 85%. Hence, in EA.hy926 cells, TG-induced Ca 2+ entry results from the activation of the STIM1/Orai1 machinery, and from the activation of TRPC3.

Cellular mechanisms underlying nitric oxide-induced vasodilation of descending vasa recta

It has been observed that vasoactivity of explanted descending vasa recta (DVR) is modulated by intrinsic nitric oxide (NO) and superoxide (O(2)(-)) production (Cao C, Edwards A, Sendeski M, Lee-Kwon W, Cui L, Cai CY, Patzak A, Pallone TL. Am J Physiol Renal Physiol 299: F1056-F1064, 2010). To elucidate the cellular mechanisms by which NO, O(2)(-) and hydrogen peroxide (H(2)O(2)) modulate DVR pericyte cytosolic Ca(2+) concentration ([Ca](cyt)) and vasoactivity, we expanded our mathematical model of Ca(2+) signaling in pericytes. We incorporated simulations of the pathways that translate an increase in [Ca](cyt) to the activation of myosin light chain (MLC) kinase and cell contraction, as well as the kinetics of NO and reactive oxygen species formation and their effects on [Ca](cyt) and MLC phosphorylation. The model reproduced experimentally observed trends of DVR vasoactivity that accompany exposure to N(ω)-nitro-L-arginine methyl ester, 8-Br-cGMP, Tempol, and H(2)O(2). Our results suggest that under resting conditions, NO-induced activation of cGMP maintains low levels of [Ca](cyt) and MLC phosphorylation to minimize basal tone. This results from stimulation of Ca(2+) uptake from the cytosol into the SR via SERCA pumps, Ca(2+) efflux into the extracellular space via plasma membrane Ca(2+) pumps, and MLC phosphatase (MLCP) activity. We predict that basal concentrations of O(2)(-) and H(2)O(2) have negligible effects on Ca(2+) signaling and MLC phosphorylation. At concentrations above 1 nM, O(2)(-) is predicted to modulate [Ca(cyt)] and MCLP activity mostly by reducing NO bioavailability. The DVR vasoconstriction that is induced by high concentrations of H(2)O(2) can be explained by H(2)O(2)-mediated downregulation of MLCP and SERCA activity. We conclude that intrinsic generation of NO by the DVR wall may be sufficient to inhibit vasoconstriction by maintaining suppression of MLC phosphorylation.

Modeling CICR in rat ventricular myocytes: voltage clamp studies

BackgroundThe past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca2+ loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca2+ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca2+ concentration ([Ca2+]myo).MethodsThe cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed Ca2+ channels (trigger-channel and release-channel). It releases Ca2+ flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[Ca2+]myo.ResultsOur model reproduces measured VC data published by several laboratories, and generates graded Ca2+ release at high Ca2+ gain in a homeostatically-controlled environment where [Ca2+]myo is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR Ca2+ release, its activation by trigger Ca2+, and its refractory characteristics mediated by the luminal SR Ca2+ sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence Ca2+ homeostasis.ConclusionsWe examine the role of the above Ca2+ regulating mechanisms in handling various types of induced disturbances in Ca2+ levels by quantifying cellular Ca2+ balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses.