Dynamics of Calcium Fluxes in Nonexcitable Cells: Mathematical Modeling

Mathematical models simulating the dynamics of calcium redistribution (elicited by experimental interference with the pathways of calcium fluxes) in cellular compartments have been developed, based on a minimal scheme of the pathways of calcium fluxes in nonexcitable cells suspended in calcium-free medium. The models are consistent with available experimental data. All parameters are quantitatively related to the intrinsic properties of calcium adenosine triphosphatases (ATPases) and cellular membranes; there is no interdependence between the parameters. The models can be used as the basis for quantitative analysis and interpretation of experimental data. The activities of plasma membrane and sarcoendoplasmic reticulum calcium ATPases (PMCA and SERCAs) are governed by different mechanisms. PMCA is likely to undergo transitions from inactive to active to “dormant” (not identical to the initial) and back to inactive states, the mean duration of the cycle lasting for minutes or longer. The sequence of the transitions is initiated, presumably, by an increase in cytosolic calcium concentration. The transition of PMCA from inactive to active (at least at low rates of increase in cytosolic calcium concentration) is likely to be slower than that from active to dormant. SERCA, presumably, transits from inactive to active state in response to increases in calcium leakage from calcium stores. Whereas PMCA extrudes excess calcium (a definite quantity of it) in a short pulse, SERCA retakes calcium back into the stores permanently at a high rate. The models presented here may be the best means for the moment to quantitatively relate the dynamics of calcium fluxes in nonexcitable cells with known or putative properties of the mechanisms underlying activation of calcium ATPases.     

Plasma membrane calcium pump and sodium-calcium exchanger in maintenance and control of calcium concentrations in platelets

The purpose of this research was to elucidate the activity of the mechanisms responsible for control of cytosolic calcium concentration in platelets by modeling the time-course of the concentration changing in response to discharge of the intracellular stores or store-operated calcium entry (SOCE). The parameters estimated as a result of model fitting to experimental data are related to physiological or pathological state of the cells. It has been shown that: (a) the time-course is determined by the passive calcium fluxes and activities of the corresponding mechanisms; (b) the decline in the concentration (after its rise) develops due to activity of plasma membrane calcium ATPase (PMCA) both in the case of discharge of the stores of platelets contained in calcium-free medium and in the case of SOCE; (c) impulsive extrusion of calcium in response to its sudden influx, presumably, is the main function of PMCA; (d) the function of sodium-calcium exchanger (NCX) is to extrude calcium excess by permanent counteracting its influx.     

Collaborative effect of SERCA and PMCA in cytosolic calcium homeostasis in human platelets

Intracellular free Ca2+ concentration ([Ca2+]c) is finely regulated by several mechanisms that either increase or reduce [Ca2+]c. Two different Ca2+ pumps have been described so far as the main mechanisms for Ca2+ removal from the cytosol, either by its sequestration into the stores, mediated by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) or by Ca2+ extrusion to the extracellular medium, by the plasma membrane Ca2+-ATPase (PMCA). We have used inhibitors of these pumps to analyze their Ca2+ clearance efficacy in human platelets stimulated by the physiological agonist thrombin. Results demonstrate that, after platelet stimulation with thrombin, activation of SERCA precedes that of PMCA, although the ability of PMCA to remove Ca2+ from the cytosol last longer than that of SERCA. The efficacy of SERCA and PMCA removing Ca2+ from the cytosol is reduced when the concentration of thrombin increases. This phenomenon correlates with the greater increase in [Ca2+]c induced by higher concentrations of thrombin, which further confirms that SERCA and PMCA activities are regulated by [Ca2+]c.     

The Novel PMCA2 Pump Mutation Tommy Impairs Cytosolic Calcium Clearance in Hair Cells and Links to Deafness in Mice

The mechanotransduction process in hair cells in the inner ear is associated with the influx of calcium from the endolymph. Calcium is exported back to the endolymph via the splice variant w/a of the PMCA2 of the stereocilia membrane. To further investigate the role of the pump, we have identified and characterized a novel ENU-induced mouse mutation, Tommy, in the PMCA2 gene. The mutation causes a non-conservative E629K change in the second intracellular loop of the pump that harbors the active site. Tommy mice show profound hearing impairment from P18, with significant differences in hearing thresholds between wild type and heterozygotes. Expression of mutant PMCA2 in CHO cells shows calcium extrusion impairment; specifically, the long term, non-stimulated calcium extrusion activity of the pump is inhibited. Calcium extrusion was investigated directly in neonatal organotypic cultures of the utricle sensory epithelium in Tommy mice. Confocal imaging combined with flash photolysis of caged calcium showed impairment of calcium export in both Tommy heterozygotes and homozygotes. Immunofluorescence studies of the organ of Corti in homozygous Tommy mice showed a progressive base to apex degeneration of hair cells after P40. Our results on the Tommy mutation along with previously observed interactions between cadherin-23 and PMCA2 mutations in mouse and humans underline the importance of maintaining the appropriate calcium concentrations in the endolymph to control the rigidity of cadherin and ensure the function of interstereocilia links, including tip links, of the stereocilia bundle.     

Effects of SERCA and PMCA inhibitors on the survival of rat cochlear hair cells during ischemia in vitro

An important mechanism underlying cochlear hair cell (HC) susceptibility to hypoxia/ischemia is the influx of Ca(2+). Two main ATP-dependent mechanisms contribute to maintaining low Ca(2+) levels: uptake of Ca(2+) into intracellular stores via smooth endoplasmic reticulum calcium ATPase (SERCA) and extrusion of Ca(2+) via plasma membrane calcium ATPase (PMCA). The effects of the SERCA inhibitors thapsigargin (10 nM-10 microM) and cyclopiazonic acid (CPA; 10-50 microM) and of the PMCA blockers eosin (1.5-10 microM) and o-vanadate (1-5 mM) on inner and outer hair cells (IHCs/OHCs) were examined in normoxia and ischemia using an in vitro model of the newborn rat cochlea. Exposure of the cultures to ischemia resulted in a significant loss of HCs. Thapsigargin and CPA had no effect. Eosin decreased the numbers of IHCs and OHCs by up to 25 % in normoxia and significantly aggravated the ischemia-induced damage to IHCs at 5 and 10 microM and to OHCs at 10 microM. o-Vanadate had no effect on IHC and OHC counts in normoxia, but aggravated the ischemia-induced HC loss in a dose-dependent manner. The effects of eosin and o-vanadate indicate that PMCA has an important role to play in protecting the HCs from ischemic cell death.     

SERCA2b and 3 play a regulatory role in store-operated calcium entry in human platelets

Two agonist-releasable Ca(2+)stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms to thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved in store-operated Ca(2+)entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca(2+)-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca(2+)content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca(2+)in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 microM TBHQ, individually or simultaneously, accelerated Ca(2+) store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition, TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca(2+) influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca(2+) extrusion by PMCA or the Na(+)/Ca(2+) exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1 channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets.     

Physical properties of two types of calcium stores and SERCAs in human platelets

The aim of this work was to obtain experimental data depending on the properties of calcium stores and SERCAs, to analyse these data in terms of the models based on simulation of the cellular compartments as communicating vessels, and to relate this way the data to the above properties. The main characteristics of the stores and corresponding SERCAs have been estimated. Calcium content in the DTS is ∼1.5 × 10⁶ ions per cell, that in the acidic stores, ∼0.64 × 10⁶ ions per cell. The rate constant of background calcium leakage from the DTS is ∼0.0033 s⁻¹, that from the acidic stores, ∼0.1 s⁻¹. The background activity of SERCA2b is ∼0.22 × 10⁶ s⁻¹ ions per cell, that of SERCA3, ∼2.5 × 10⁶ s⁻¹ ions per cell. The properties of both calcium stores and the SERCAs and the characteristics found might be related to physiological or pathological state of the cells.     

Calcium fluxes into and out of cytosol in human platelets: analysis of experimental data

The purpose of this research was to analyse experimental data concerning cytosolic calcium concentration in view of the mechanisms involved in calcium fluxes in human platelets. The parameters of model curves are related to the properties of the entities responsible for control or maintenance of cytosolic calcium concentration. It has been shown that: (a) biphasicity of increase in cytosolic calcium concentration caused by inhibition of SERCAs either by TBHQ and TG or by TG alone is related to fast and slow discharge of acidic calcium stores and DTS; (b) biphasicity of decline in cytosolic calcium concentration after its rise caused by stimulation of platelets by the agonists is related to non-synchronous extrusion of calcium by PMCA and NCX; (c) NCX is active only in calcium containing medium: calcium ion(s) are necessary to be bound to the site(s) located on the medium-facing side of the (macro)molecule; (d) PMCA is likely to be activated either by binding calcium ion(s) to the site(s) located on its cytosol-facing side or by unbinding identical ion(s) from the site(s) on its medium-facing side.     

Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle

Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.     

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.     

Role of endothelial Ca2+ stores in the regulation of hydraulic conductivity of Rana microvessels in vivo

Vascular permeability is regulated by endothelial cytosolic Ca(2+) concentration ([Ca(2+)](i)). To determine whether vascular permeability is dependent on extracellular Ca(2+) influx or release of Ca(2+) from stores, hydraulic conductivity (L(p)) was measured in single perfused frog mesenteric microvessels in the presence and absence of Ca(2+) influx and store depletion. Prevention of Ca(2+) reuptake into stores by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibition increased L(p) in the absence of extracellular Ca(2+) influx. L(p) was further increased when Ca(2+) influx was restored. Depletion of the Ca(2+) stores with ionomycin and SERCA inhibition increased L(p) in the presence and the absence of extracellular Ca(2+) influx. However, store depletion in itself did not significantly increase L(p) in the absence of active Ca(2+) release from stores into the cytoplasm. There was a significant positive correlation between baseline permeability and the magnitude of the responses to both Ca(2+) store release and Ca(2+) influx, indicating that the Ca(2+) regulating properties of the endothelial cells may regulate the baseline L(p). To investigate the role of Ca(2+) stores in regulation of L(p), the relationship between SERCA inhibition and store release was studied. The magnitude of the L(p) increase during SERCA inhibition significantly and inversely correlated with that during store release by Ca(2+) ionophore, implying that the degree of store depletion regulates the size of the increase on L(p). These data show that microvascular permeability in vivo can be increased by agents that release Ca(2+) from stores in the absence of Ca(2+) influx. They also show that capacitative Ca(2+) entry results in increased L(p) and that the size of the permeability increase can be regulated by the degree of Ca(2+) release.     

The effect of calcium pump inhibitors on the response of intracellular calcium to caffeine in snail neurones

We have measured intracellular free calcium ([Ca(2+)]i) using Fura-2 or Ca(2+)-sensitive microelectrodes in voltage-clamped neurones of the snail, Helix aspersa. Caffeine-induced transient increases in [Ca(2+)]i were normally followed by a brief fall of [Ca(2+)]i below its pre-caffeine level. We investigated the cause of this undershoot by raising [Ca(2+)]i; and by inhibiting the plasma membrane or endoplasmic reticulum Ca ATPases (PMCA or SERCA respectively). When the cell membrane potential was decreased from -60 to -25mV, steady-state [Ca(2+)]i increased. The caffeine-induced transients were smaller while the undershoots were larger than in control conditions. When the PMCA was inhibited by high pH the steady-state [Ca(2+)]i increased by 100-400nM. The caffeine-induced [Ca(2+)]i increase and the subsequent undershoot both became larger. Injection of orthovanadate, which inhibits the PMCA and increases [Ca(2+)]i, did not block either effect of caffeine. But when the SERCA was inhibited by cyclopiazonic acid the undershoot disappeared. The phosphodiesterase inhibitor IBMX did not influence the undershoot. These results suggest that the undershoot is generated by the Ca(2+)] ATPase of the stores rather than that of the plasma membrane. Since the undershoot increased as [Ca(2+)]i increased, we conclude that at higher levels of [Ca(2+)]i the stores refill more rapidly.     

The PLEKHA7–PDZD11 complex regulates the localization of the calcium pump PMCA and calcium handling in cultured cells

The plasma membrane calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adapter protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here, we test this hypothesis using cultured cell model systems. We show using immunofluorescence microscopy and a surface biotinylation assay that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct epithelial cells results in increased accumulation of endogenous PMCA at lateral cell–cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, coexpression of PDZD11 reduces membrane accumulation of overexpressed PMCA4x/b, and analysis of cytosolic calcium transients shows that PDZD11 counteracts calcium extrusion activity of overexpressed PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mouse kidney collecting duct) cells increases the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7–PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA.     

Reaction diffusion modeling of calcium dynamics with realistic ER geometry

We describe a finite-element model of mast cell calcium dynamics that incorporates the endoplasmic reticulum's complex geometry. The model is built upon a three-dimensional reconstruction of the endoplasmic reticulum (ER) from an electron tomographic tilt series. Tetrahedral meshes provide volumetric representations of the ER lumen, ER membrane, cytoplasm, and plasma membrane. The reaction-diffusion model simultaneously tracks changes in cytoplasmic and ER intraluminal calcium concentrations and includes luminal and cytoplasmic protein buffers. Transport fluxes via PMCA, SERCA, ER leakage, and Type II IP3 receptors are also represented. Unique features of the model include stochastic behavior of IP3 receptor calcium channels and comparisons of channel open times when diffusely distributed or aggregated in clusters on the ER surface. Simulations show that IP3R channels in close proximity modulate activity of their neighbors through local Ca2+ feedback effects. Cytoplasmic calcium levels rise higher, and ER luminal calcium concentrations drop lower, after IP3-mediated release from receptors in the diffuse configuration. Simulation results also suggest that the buffering capacity of the ER, and not restricted diffusion, is the predominant factor influencing average luminal calcium concentrations.     

Sarco(endo)plasmic reticulum and plasmalemmal Ca(2+)-ATPase activities in cremaster muscles and sacs differ according to the associated inguinal pathology

Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and plasmalemmal Ca(2+)-ATPase (PMCA) activities in cremaster muscles and sacs, which have been subjected to different autonomic tonuses, were determined and compared. Samples of cremaster muscles and sacs associated with male or female inguinal hernia, hydrocele or undescended testis were obtained from children during operations and activities of SERCA and PMCA were determined. While highest SERCA and PMCA activities were encountered among cremaster muscles and sacs associated with undescended testis, least activities were encountered among structures associated with hydrocele. The alterations in SERCA and PMCA activities in cremaster muscles associated with undescended testis appear to reflect the attempts at maintaining the levels of cytosolic calcium. Despite similar total calcium contents, lower SERCA and PMCA activities were found in sacs associated with hydrocele compared to those associated with undescended testis suggest a difference among the levels of cytosolic calcium.     

Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

Plasma membrane Ca²⁺-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca²⁺]_i). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca²⁺]_i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.     

Role of Ca2+,Mg2+-ATPases in Diabetes-Induced Alterations in Calcium Homeostasis in Input Neurons of the Nociceptive System

In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca²⁺ in input neurons of the nociceptive system increases, Ca²⁺ signals are prolonged, while Ca²⁺ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca²⁺,Mg²⁺-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca²⁺,Mg²⁺-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca²⁺,Mg²⁺-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P < 0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P < 0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca²⁺ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities.     

Role of proton gradients and vacuola H(+)-ATPases in the refilling of intracellular calcium stores in exocrine cells

Numerous hormones and neurotransmitters activate cells by increasing cytosolic calcium concentration ([Ca(2+)](i)), a key regulatory factor for many cellular processes. A pivotal feature of these Ca(2+) signals is the release of Ca(2+) from intracellular stores, which is followed by activation of extracellular calcium influx, allowing refilling of the stores by SERCA pumps associated with the endoplasmic reticulum. Although the mechanisms of calcium release and calcium influx have been extensively studied, the biology of the Ca(2+) stores is poorly understood. The presence of heterogeneous calcium pools in cells has been previously reported [1] [2] [3]. Although recent technical improvements have confirmed this heterogeneity [4], knowledge about the mechanisms underlying Ca(2+) transport within the stores is very scarce and rather speculative. A recent study in polarized exocrine cells [5] has revealed the existence of Ca(2+) tunneling from basolateral stores to luminal pools, where Ca(2+) is initially released upon cell activation. Here, we present evidence that, during stimulation, Ca(2+) transported into basolateral stores by SERCA pumps is conveyed toward the luminal pools driven by proton gradients generated by vacuolar H(+)-ATPases. This finding unveils a new aspect of the machinery of Ca(2+) stores.