Basal Calcium Concentration Research Articles

Abstract P1094: Sexual Dimorphism In Histone Deacetylases Activity Regulates Ventricular Calcium Handling Properties

Introduction: There is a sexual dimorphism in cardiac excitation contraction coupling (ECC) with female ventricular myocytes (F VM ) exhibiting an attenuated basal calcium concentration ([Ca] i ), Ca transient (CaT) amplitude, and a prolonged CaT decay constant (τ). Increased expression levels of the sodium-calcium exchanger (NCX) have been described in F VM however, the role NCX expression plays in sexual dimorphism of physiological and pathophysiological Ca handling properties, and how NCX expression is regulated in a sex specific manner remains to be determined. Here we tested the hypothesis, that a sexual dimorphism in HDAC activity regulates NCX expression and NCXs differential contribution to the CaT adds to the sexual dimorphism in ventricular ECC. Method: [Ca] i was quantified in field stimulated, freshly isolated ventricular myocytes (VMs) from male (M VM ) and female (F VM ) FVB/N mice loaded with the Ca sensitive fluorescent dye Fluo-4/AM. Changes in NCX expression were quantified by qPCR and western blotting. Results: Under basal conditions F VM exhibited an attenuated CaT amplitude (ΔF/F 0 ; M VM :1.8±0.08, F VM :1.4±0.06, p=0.038) and prolonged CaT decay constant (τ: M VM :0.37±0.01ms, F VM :0.46±0.02ms, p<0.01). The caffeine induced Ca transient (CaT caf ) amplitude was comparable in F VM and M VM , but the CaT caf decay constant (τ caf: M VM : 3.34±0.22s ; F VM :4.81±0.36s) was longer in F VM although increased NCX expression was determined on the protein and mRNA level. Consistent with increased NCX expression, F VM showed an attenuated increase in basal [Ca] i (F/F 0 ; M= 60%, F=44%, p=0.0033) and τ (M=0.37±0.01s, F=0.46±0.02s, p<0.0001) when SERCA activity was attenuated (CPA: 10μM). Also during conditions favoring NCX reverse mode ([Na] o =93, 46 mM) F VM exhibited a larger increase in CaT amplitude (ΔF/F 0 ; M=81%, F=133%, p<0.01) and an increased propensity for spontaneous Ca release events. Inhibition of HDAC class II (LMK: 5mg/kg/day) did not change Ca handling properties in F VM but slowed τ (M LMK : 0.44±0.03ms) and τ caf in M VM (M LMK =5.1±0.33s) making them comparable to F VM . Conclusion: The sexual dimorphism in ventricular ECC is mediated by attenuated HDAC activity in F VM s and facilitates NCX Ca overload.

Compensatory mechanisms in resistant Anopheles gambiae AcerKis and KdrKis neurons modulate insecticide-based mosquito control

In the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.

Examining the effects of the microglial PLCG2 P522R mutation by transplantation of human stem cell‐derived microglia in chimeric AD mice

AbstractBackgroundGenetic studies have uncovered several Alzheimer’s Disease (AD) risk genes that are highly expressed in microglia, suggesting that immune dysregulation plays a critical role in the development and progression of AD. In line with this, a recently discovered polymorphism (P522R) in the microglia‐expressed gene Phospholipase C‐Gamma 2 (PLCG2) was shown to be protective for AD. Interestingly, PLCG2 codes for a protein that regulates the release of calcium from ER stores and is also a component of the downstream signaling pathway of TREM2, another microglial AD risk gene. This underscores the need to improve our understanding of microglial calcium signaling, the effects of AD risk genes on this pathway, and the impacts of PLCG2 and calcium signaling on human microglial function.MethodsCRISPR gene editing was used to produce isogenic iPSCs that express the AD protective mutation in PLCG2. Several in vitro assays were then performed to examine calcium signaling pathways and phagocytosis. PLCG2‐P522R and WT HPCs were also transplanted directly into xenotransplantation‐compatible mouse pups (MITRG‐5xfAD mice), where they readily differentiated into human microglia, exhibiting typical markers and morphology.. Xenotransplanted human microglia (x‐MG) were then examined by confocal microscopy and isolated from the brain for single‐cell RNA sequencing.ResultsThus far our in vitro studies suggest that the P522R coding mutation increases both basal cytosolic calcium concentrations and amyloid phagocytosis. Additional functional experiments are further examining the interactions between PLCG2 mutations and calcium signaling during the uptake of AD‐relevant substrates. In addition, single cell RNA sequencing studies of chimeric mice reveal that the P522R mutation leads to an increased percentage of human microglia that have adopted a disease‐associated microglial (DAM) phenotype in vivo.ConclusionsBy combining iPSC‐microglia, CRISPR gene editing, and chimeric AD mice these studies have begun to uncover the impact of PLCG2 mutations on human microglial function and reveal that the protective P522R mutation increases Ab phagocytosis and promotes the adoption of a DAM‐like microglial response.

Knockdown of Letm1 Protein Affects Mitochondrial Calcium Homeostasis and Cell Viability in Trypanosoma cruzi

Calcium ion (Ca2+) is an important second messenger in the life cycle of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease. It is well established that mitochondria have a central role in intracellular Ca2+ homeostasis. While the mechanisms of mitochondrial Ca2+ release in T. cruzi are not completely understood, it has been proposed that Letm1 (Leucine Zipper and EF‐Hand Containing Transmembrane Protein 1), a protein localized to the inner mitochondrial membrane, mediates the extrusion of Ca2+ from mitochondria via a Ca2+/H+ exchange mechanism. Using the CRISPR/Cas9 system we generated TcLetm1 knockdown (TcLetm1‐KD) cell lines. By electroporation of the parasite in presence of pTREX‐Letm1 plasmid, we also obtained a parasite line overexpressing Letm1 protein (OE‐Letm1). Using control parasites for knockdown (transfected with a scrambled sgRNA ‐ SCR) or overexpressing Letm1 protein (pTREX‐n) we have performed cell biology and biochemical experiments to assess the function of Letm1 in T. cruzi. We found that, although overexpression of Letm1 has no effect in epimastigotes proliferation, TcLetm1‐KD epimastigotes have a significantly lower proliferation rate. The ongoing biochemical experiments suggest that the protein Letm1 is, indeed, involved in mitochondrial Ca2+ homeostasis. Using digitonin permeabilized cells, we observed that, while overexpression of Letm1 increases the rates of both net mitochondrial Ca2+ uptake and release, TcLetm1‐KD has the opposite effect. Additionally, we found that TcLetm1‐KD parasites showed an increase in basal mitochondrial calcium concentration. In cell respiration experiments, TcLetm1‐KD cells showed lower oxygen consumption rates than control cells and increased ADP:ATP ratio. Knockdown and overexpression of Letm1 were achieved in T. cruzi for the first time and these cells are being used to study the activity of TcLetm1 as a mitochondrial Ca2+/H+ exchanger.Support or Funding InformationFAPESP, CNPq, and CAPES.

Multiparameter imaging of calcium and abscisic acid and high-resolution quantitative calcium measurements using R-GECO1-mTurquoise in Arabidopsis.

Calcium signals occur in specific spatio-temporal patterns in response to various stimuli and are coordinated with, for example, hormonal signals, for physiological and developmental adaptations. Quantification of calcium together with other signalling molecules is required for correlative analyses and to decipher downstream calcium-decoding mechanisms. Simultaneous invivo imaging of calcium and abscisic acid has been performed here to investigate the interdependence of the respective signalling processes in Arabidopsis thaliana roots. Advanced ratiometric genetically encoded calcium indicators have been generated and invivo calcium calibration protocols were established to determine absolute calcium concentration changes in response to auxin and ATP. In roots, abscisic acid induced long-term basal calcium concentration increases, while auxin triggered rapid signals in the elongation zone. The advanced ratiometric calcium indicator R-GECO1-mTurquoise exhibited an increased calcium signal resolution compared to commonly used Förster resonance energy transfer-based indicators. Quantitative calcium measurements in Arabidopsis root tips using R-GECO1-mTurquoise revealed detailed maps of absolute calcium concentration changes in response to auxin and ATP. Calcium calibration protocols using R-GECO1-mTurquoise enabled high-resolution quantitative imaging of resting cytosolic calcium concentrations and their dynamic changes that revealed distinct hormonal and ATP responses in roots.

Abstract 6: Restoration of Impaired Diastolic Function in Hypertrophic Cardiomyopathy Induced Pluripotent Stem Cell-derived Cardiomyocytes by Re-balancing the Calcium Homeostasis

Background: Diastolic dysfunction is commonly seen in hypertrophic cardiomyopathy (HCM). However, the cellular mechanism is not fully understood, and no effective treatment so far has been developed. We hypothesize here that HCM patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can recapitulate the cellular mechanism, and provide us a platform for mechanistic study and for drug screening of diastolic dysfunctions in HCM. Methods and Results: We generated beating iPSC-CMs from healthy individuals and HCM patients carrying familial mutations (MYH7 R663H (n=2 lines) and MYBPC3 R943ter (n=2 lines)). Sarcomere shortening measurement in patterned iPSC-CMs with live cell confocal imaging showed significantly prolonged diastolic phase and slower relaxation velocity in HCM iPSC-CMs compared to WT cells. To elucidate the cellular mechanism, Fura-2 AM ratiometric calcium imaging showed marked elevation of resting calcium level and increased abnormal calcium handlings in HCM iPSC-CMs, which were exaggerated by β-adrenergic activation with isoproterenol. By applying calcium transient and contractile force simultaneous recording, we defined a “risk index of diastolic dysfunction” (measured as transient-contraction gain factor), which was significantly increased in HCM iPSC-CMs. Thus, both elevated basal calcium level and increased calcium sensitivity of myofilament contribute to the abnormal diastolic function in HCM iPSC-CMs. Gene expression profiling of HCM and WT iPSC-CMs indicated that increased calcium channels may underlie the increased basal calcium concentration in HCM cells. Indeed, partially blocking the calcium influx by calcium blockers reset the basal calcium level, attenuated calcium mishandling, and restored the diastolic function in HCM iPSC-CMs. Moreover, re-balancing calcium homeostasis significantly improved long-term survival rate of HCM iPSC-CMs at both basal level and under β-adrenergic stress. Conclusion: The iPSC-CM models carrying patient-specific HCM mutations recapitulated diastolic dysfunction on single cell level. Future studies using these platform may reveal additional novel cellular mechanisms and therapeutic targets of diastolic dysfunction in HCM disease.

Extracellular cGMP Modulates Learning Biphasically by Modulating Glycine Receptors, CaMKII and Glutamate-Nitric Oxide-cGMP Pathway.

It has been proposed that extracellular cGMP modulates the ability to learn a Y maze task, but the underlying mechanisms remained unknown. Here we show that extracellular cGMP, at physiological concentrations, modulates learning in the Y maze in a biphasic way by modulating the glutamate-nitric oxide-cGMP pathway in cerebellum. Extracellular cGMP reduces glycine receptors activation inducing a voltage-dependent calcium-channels-mediated increase of calcium in Purkinje neurons. This calcium increase modulates CaMKII phosphorylation in a biphasic way. When basal calcium concentration is low extracellular cGMP reduces CaMKII phosphorylation, increasing nitric oxide synthase activity, the glutamate-NO-cGMP pathway function and learning ability. When basal calcium is normal extracellular cGMP increases CaMKII phosphorylation, reducing nitric oxide synthase activity, the pathway function and learning. These data unveil new mechanisms modulating learning in the Y maze and likely other learning types which may be therapeutic targets to improve learning in pathological situations associated with altered cGMP levels.

Involvement of actin microfilament in regulation of pacemaking activity increased by hypotonic stress in cultured ICCs of murine intestine.

Distension is a regular mechanical stimulus in gastrointestinal (GI) tract. This study was designed to investigate the effect of hypotonic stress on pacemaking activity and determine whether actin microfilament is involved in its mechanism in cultured murine intestinal interstitial cells of Cajal (ICCs) by using whole-cell patch-clamp and calcium imaging techniques. Hypotonic stress induced sustained inward holding current from the baseline to -650+/-110 pA and significantly decreased amplitudes of pacemaker current. Hypotonic stress increased the intensity of basal fluorescence ratio (F/F0) from baseline to 1.09+/-0.03 and significantly increased Ca(2+) oscillation amplitude. Cytochalasin-B (20 microM), a disruptor of actin microfilaments, significantly suppressed the amplitudes of pacemaker currents and calcium oscillations, respectively. Cytochalasin-B also blocked hypotonic stress-induced sustained inward holding current and hypotonic stress-induced increase of calcium oscillations. Phalloidin (20 microM), a stabilizer of actin microfilaments, significantly enhanced the amplitudes of pacemaker currents and calcium oscillations, respectively. Despite the presence of phalloidin, hypotonic stress was still able to induce an inward holding current and increased the basal fluorescence intensity. These results suggest that hypotonic stress induces sustained inward holding current via actin microfilaments and the process is mediated by alteration of intracellular basal calcium concentration and calcium oscillation in cultured intestinal ICCs.

TRPC6 participates in the regulation of cytosolic basal calcium concentration in murine resting platelets

Cytosolic-free Ca2+ plays a crucial role in blood platelet function and is essential for thrombosis and hemostasis. Therefore, cytosolic-free Ca2+ concentration is tightly regulated in this cell. TRPC6 is expressed in platelets, and an important role for this Ca2+ channel in Ca2+ homeostasis has been reported in other cell types. The aim of this work is to study the function of TRPC6 in platelet Ca2+ homeostasis. The absence of TRPC6 resulted in an 18.73% decreased basal [Ca2+]c in resting platelets as compared to control cells. Further analysis confirmed a similar Ca2+ accumulation in wild-type and TRPC6-deficient mice; however, passive Ca2+ leak rates from agonist-sensitive intracellular stores were significantly decreased in TRPC6-deficient platelets. Biotinylation studies indicated the presence of an intracellular TRPC6 population, and subcellular fractionation indicated their presence on endoplasmic reticulum membranes. Moreover, the presence of intracellular calcium release in platelets stimulated with 1-oleoyl-2-acetyl-sn-glycerol further suggested a functional TRPC6 population located on the intracellular membranes surrounding calcium stores. However, coimmunoprecipitation assay confirmed the absence of STIM1–TRPC6 interactions in resting conditions. This findings together with the absence of extracellular Mn2+ entry in resting wild-type platelets indicate that the plasma membrane TRPC6 fraction does not play a significant role in the maintenance of basal [Ca2+]c in mouse platelets. Our results suggest an active participation of the intracellular TRPC6 fraction as a regulator of basal [Ca2+]c, controlling the passive Ca2+ leak rate from agonist-sensitive intracellular Ca2+ stores in resting platelets.

Calcium/Calmodulin-Dependent Protein Kinase Is Negatively and Positively Regulated by Calcium, Providing a Mechanism for Decoding Calcium Responses during Symbiosis Signaling

The establishment of symbiotic associations in plants requires calcium oscillations that must be decoded to invoke downstream developmental programs. In animal systems, comparable calcium oscillations are decoded by calmodulin (CaM)-dependent protein kinases, but symbiotic signaling involves a calcium/CaM-dependent protein kinase (CCaMK) that is unique to plants. CCaMK differs from the animal CaM kinases by its dual ability to bind free calcium, via calcium binding EF-hand domains on the protein, or to bind calcium complexed with CaM, via a CaM binding domain. In this study, we dissect this dual regulation of CCaMK by calcium. We find that calcium binding to the EF-hand domains promotes autophosphorylation, which negatively regulates CCaMK by stabilizing the inactive state of the protein. By contrast, calcium-dependent CaM binding overrides the effects of autophosphorylation and activates the protein. The differential calcium binding affinities of the EF-hand domains compared with those of CaM suggest that CCaMK is maintained in the inactive state at basal calcium concentrations and is activated via CaM binding during calcium oscillations. This work provides a model for decoding calcium oscillations that uses differential calcium binding affinities to create a robust molecular switch that is responsive to calcium concentrations associated with both the basal state and with oscillations.

Кінетичні закономірності дії калікс[4]арену С-90 мембрани та на концентрацію Са2+ в незбуджених клітинах міометрія

Plasma membrane Ca2+,Mg2+-ATPase is an important element of general myometrium tonus control mechanism, which also makes a contribution to muscle tension relaxation after its contraction. Expiriments were done on the myometrial cell plasma membrane suspension, which was treated with 0.1% digitonin solution. The authors have investigated the inhibitory action of calix[4]arene C-90 (5,11,17,23-tetra(trifluor) methyl(phenylsulphonylimino)-methylamino-25,26,27,28-tetra propoxi-calix[4]arene) on the Ca2+,Mg2+-ATPase activity (the magnitude of 10.5 was 20.2 +/- 0.5 mkM). The inhibitory action of calix[4]arene C-90 on the activity of Ca2+, Mg2+-ATPase is explained as cooperative action of four trifluormethyl(phenylsulfonylimino)methylamino groups that are spatially oriented on the calix[4]-arene base rather than with the action of tetra-phenol macrocycle or separate pharmacophore sulphonilamidin groups. Considering established kinetic pattern of calix[4]arene C-90 inhibitory action on the plasma membrane Ca2+,Mg2+-ATPase activity, stationary kinetical model of basal calcium concentration control in unexcited uterus myocytes was developed. It is assumed that obtained results may be promising for creation of new generation ("supramolecular") pharmacological agent - uterus basal tonus stimulator - on the base of calix[4] arene C-90.

Transient Receptor Potential Channel 1 (TRPC1) Reduces Calcium Permeability in Heteromeric Channel Complexes

Specific biological roles of the classical transient receptor potential channel 1 (TRPC1) are still largely elusive. To investigate the function of TRPC1 proteins in cell physiology, we studied heterologously expressed TRPC1 channels and found that recombinant TRPC1 subunits do not form functional homomeric channels. Instead, by electrophysiological analysis TRPC1 was shown to form functional heteromeric, receptor-operated channel complexes with TRPC3, -4, -5, -6, and -7 indicating that TRPC1 proteins can co-assemble with all members of the TRPC subfamily. In all TRPC1-containing heteromers, TRPC1 subunits significantly decreased calcium permeation. The exchange of select amino acids in the putative pore-forming region of TRPC1 further reduced calcium permeability, suggesting that TRPC1 subunits contribute to the channel pore. In immortalized immature gonadotropin-releasing hormone neurons endogenously expressing TRPC1, -2, -5, and -6, down-regulation of TRPC1 resulted in increased calcium permeability and elevated basal cytosolic calcium concentrations. We did not observe any involvement of TRPC1 in store-operated cation influx. Notably, TRPC1 suppressed the migration of gonadotropin-releasing hormone neurons without affecting cell proliferation. Conversely, in TRPC1 knockdown neurons, specific migratory properties like distance covered, locomotion speed, and directionality were increased. These findings suggest a novel regulatory mechanism relying on the expression of TRPC1 and the subsequent formation of heteromeric TRPC channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration.

The Small G Protein Rac1 Activates Phospholipase Cδ1 through Phospholipase Cβ2

Rac1, which is associated with cytoskeletal pathways, can activate phospholipase Cbeta2 (PLCbeta2) to increase intracellular Ca(2+) levels. This increased Ca(2+) can in turn activate the very robust PLCdelta1 to synergize Ca(2+) signals. We have previously found that PLCbeta2 will bind to and inhibit PLCdelta1 in solution by an unknown mechanism and that PLCbeta2.PLCdelta1 complexes can be disrupted by Gbetagamma subunits. However, because the major populations of PLCbeta2 and PLCdelta1 are cytosolic, their regulation by Gbetagamma subunits is not clear. Here, we have found that the pleckstrin homology (PH) domains of PLCbeta2 and PLCbeta3 are the regions that result in PLCdelta1 binding and inhibition. In cells, PLCbeta2.PLCdelta1 form complexes as seen by Förster resonance energy transfer and co-immunoprecipitation, and microinjection of PHbeta2 dissociates the complex. Using PHbeta2 as a tool to assess the contribution of PLCbeta inhibition of PLCdelta1 to Ca(2+) release, we found that, although PHbeta2 only results in a 25% inhibition of PLCdelta1 in solution, in cells the presence of PHbeta2 appears to eliminates Ca(2+) release suggesting a large threshold effect. We found that the small plasma membrane population of PLCbeta2.PLCdelta1 is disrupted by activation of heterotrimeric G proteins, and that the major cytosolic population of the complexes are disrupted by Rac1 activation. Thus, the activity of PLCdelta1 is controlled by the amount of bound PLCbeta2 that changes with displacement of the enzyme by heterotrimeric or small G proteins. Through PLCbeta2, PLCdelta1 activation is linked to surface receptors as well as signals that mediate cytoskeletal pathways.

Presynaptic Miniature Gabaergic Currents in Developing Interneurons

Miniature synaptic currents have long been known to represent random transmitter release under resting conditions, but much remains to be learned about their nature and function in central synapses. In this work, we describe a new class of miniature currents ("preminis") that arise by the autocrine activation of axonal receptors following random vesicular release. Preminis are prominent in gabaergic synapses made by cerebellar interneurons during the development of the molecular layer. Unlike ordinary miniature postsynaptic currents in the same cells, premini frequencies are strongly enhanced by subthreshold depolarization, suggesting that the membrane depolarization they produce belongs to a feedback loop regulating neurotransmitter release. Thus, preminis could guide the formation of the interneuron network by enhancing neurotransmitter release at recently formed synaptic contacts.

Calcium Ion Gradients and Dynamics in Cultured Skin Slices of Rat Hindpaw in Response to Stimulation with ATP

Ionotropic receptors, originally found in the brain, were recently also identified in epidermal keratinocytes. Moreover, concentration gradients and movement of calcium are crucial in epidermal homeostasis. Thus, imaging of calcium in the living epidermis is expected to provide insight into epidermal physiology and pathology. Here we describe the imaging of calcium dynamics in the living epidermis of cultured skin slices. The basal calcium concentration was highest in the upper layer of the epidermis. The increase of intracellular calcium in response to adenosine triphosphate (ATP) varied in each layer of epidermis, and was greater at the bottom than in the uppermost layer. Further, the extent of elevation of intracellular calcium in response to ATP in cultured keratinocytes varied depending on the level of differentiation. These results suggest that the response to stimulation of keratinocytes in cultured skin slices varies depending upon the location (depth) within the epidermis.

A STIM for Resting Cells

One of the "Breakthroughs of the Year" noted in last year’s compendium at STKE was the discovery of the role of STIM proteins, particularly STIM1, in coupling stimulus-induced depletion of intracellular calcium stores to influx of extracellular calcium through the plasma membrane calcium channel Orai1. Brandman et al . have now capped off 2007 with a paper describing a key role of STIM2 in regulation of basal concentrations of calcium in the cytoplasm and endoplasmic reticulum (ER) in unstimulated cells. The authors used an siRNA screen to find proteins that regulated basal calcium concentrations in cultured human HeLa cells. They sensitized the cells to depletion of calcium regulatory proteins by exposing them to low or high concentrations of extracellular calcium for 24 hours. The protein that stood out as a positive regulator of basal calcium concentrations was STIM2. Automated low-magnification imaging of fluorescently tagged STIM2 showed that, like STIM1, the protein translocated to the plasma membrane when intracellular stores of calcium were depleted. However, STIM2 was sensitive to smaller changes in the concentration of calcium in the ER and seemed to be tuned to provide a linear response to small changes in basal concentrations of calcium, whereas STIM1 has a switchlike response to large changes in the concentration of calcium after receptor stimulation. Thus, the authors propose that STIM2 may help maintain a constant basal concentration of calcium in the cytoplasm and ER, thus avoiding accidental activation of calcium signaling pathways. STIM2 may also provide a way for weak receptor stimuli to allow persistent changes in calcium concentrations, with STIM1 operating primarily to handle strong receptor stimuli. O. Brandman, J. Liou, W. S. Park, T. Meyer, STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca 2+ levels. Cell 131 , 1327-1339 (2007). [PubMed]

Impact of parathyroidectomy on neutrophil cytosolic calcium in chronic kidney disease patients: a prospective parallel group trial

Polymorphonuclear leucocytes (PMNs) of chronic kidney disease (CKD) patients display elevated basal cytosolic calcium concentrations (iCa(2+)). As parathyroid hormone is considered to substantially contribute to the inappropriate cellular entry of calcium in uraemia, we hypothesized that parathyroidectomy lowers PMN iCa(2+). Prospective parallel group trial at a tertiary care centre. SUBJECTS, INTERVENTION AND MAIN OUTCOME MEASURES: Two patient cohorts (cohort 1: 14 CKD patients; cohort 2: 14 renal transplant recipients) underwent parathyroidectomy for uncontrolled secondary hyperparathyroidism. We assessed PMN iCa(2+) (primary objective) spectrofluorimetrically 1 day before and 20 days after intervention (secondary objective: PMN glucose uptake). Data were compared with those of 16 matched maintenance haemodialysis patients (cohort 3), and to 15 healthy subjects (cohort 4), by generalized estimating equations. PMN iCa(2+) of cohort 1 decreased over time and was significantly higher than that of cohort 3 before but not after parathyroidectomy [mean difference before/after parathyroidectomy: 19.1 nmol L(-1) (95% confidence interval: 9.4-22.4), P =0.0003/-3.2 (-20.9-14.5), P = 0.71]. PMN iCa(2+) of cohort 2 decreased over time, but we found no significant difference in comparison with cohort 3 [mean difference before/after parathyroidectomy: 6.5 nmol L(-1) (-9.4-22.4), P = 0.4/-15.8 (-43.6-12.0), P = 0.25]. PMN iCa(2+) of all CKD patients was substantially higher in comparison with that of healthy subjects [cohort 4 vs. 3: -35.3 (-48.9-21.6), P < 0.001]. PMN glucose uptake increased significantly in both interventional cohorts in comparison with cohort 3. Parathyroidectomy lowers, but does not normalize PMN iCa(2+) of CKD patients. Further variables, possibly uraemic retention solutes, control both PMN iCa(2+) and functional responses.

Enhanced excitability of hippocampal mossy fibers and CA3 neurons under dietary zinc deficiency

On the basis of the evidence that susceptibility to kainate-induced seizures is enhanced by zinc deficiency and that glutamate concentrations in hippocampal extracellular fluid are excessively increased during seizures, excitability of hippocampal mossy fibers and CA3 neurons was examined using hippocampal slices, which were prepare from mice fed a zinc-deficient diet for 4 weeks. The spatio-temporal dynamics of zinc and calcium was monitored using their indicators, membrane-impermeable ZnAF-2 and membrane-permeable fura-2 AM, respectively. When the molecular layer of dentate gyrus was stimulated with 100 mM KCl for 1 s, the increased percentages of extracellular zinc in the stratum lucidum and CA3 pyramidal cell layer were higher in zinc-deficient mice than in the control mice, implying that glutamate release from the mossy fibers of the dentate granular cells is enhanced by zinc deficiency. Judging from the increased percentages, however, the amount of zinc released was estimated to be less in zinc-deficient mice. On the other hand, the basal calcium concentrations in the stratum lucidum and CA3 pyramidal cell layer detected with fura-2 were higher in zinc-deficient mice than in the control mice, indicating that hippocampal calcium homeostasis is affected by zinc deficiency. Furthermore, the increased percentage of intracellular calcium in the stratum lucidum by stimulation with high K + was enhanced by the zinc deficiency. The alteration of hippocampal calcium homeostasis seems to enhance excitability of dentate granular cells in zinc deficiency, following by an enhanced excitability of postsynaptic structures in CA3 neurons.

P2Y-receptors mediating an inhibition of the evoked entry of calcium through N-type calcium channels at neuronal processes.

In the search for P2-receptors modulating the stimulation-evoked entry of calcium at processes of PC12 cells differentiated in the presence of nerve growth factor and neurotrophin-3, electrically evoked increases in free calcium were assessed by fura-2 microfluorimetry. Omission of calcium and addition of cadmium (100 microM) or the N-type calcium channel blocker omega-conotoxin GVIA (0.5 microM) abolished or markedly reduced the evoked responses. The P2Y-receptor agonists 2-methylthio adenosine 5'-diphosphate (2-methylthio-ADP), ADP, and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) inhibited the electrically evoked entry of calcium without any changes in basal calcium concentrations. 2-Methylthio-ADP was the most potent agonist. Adenosine, P(1),P(4)-di(adenosine-5')-tetraphosphate (Ap4A), UDP, and UTP (30 microM each) had no effect. The effect of ADPbetaS (30 microM) was abolished by the P2-antagonists reactive blue 2 (3 microM), suramin (100 microM), 2-methylthio-AMP (10 microM), p-chloromercuriphenyl sulfonic acid (1 microM), and AR-C 69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene adenosine 5'-triphosphate] (300 nM). In contrast, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (10 microM), the selective P2Y1-receptor antagonist MRS 2179 (N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate; 10 microM), as well as the adenosine A(1)-receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 100 nM), caused no change. Pretreatment with pertussis toxin abolished the effect of ADPbetaS. Reverse transcriptase-polymerase chain reaction revealed the presence of mRNA for P2Y12-receptors in nondifferentiated and differentiated PC12 cells. The results indicate that processes of differentiated PC12 cells possess P2Y12-receptors coupling to pertussis toxin-sensitive G-proteins and mediating an inhibition of the stimulation-evoked entry of calcium through omega-conotoxin GVIA-sensitive calcium channels. This suggests a role of P2Y12-receptors in neuromodulation in addition to their involvement in platelet aggregation.

Calcium dynamics and buffering in oculomotor neurones from mouse that are particularly resistant during amyotrophic lateral sclerosis (ALS)-related motoneurone disease.

Motoneurones are particularly vulnerable both in human forms of amyotrophic lateral sclerosis (ALS) and corresponding animal models of the disease. While most motoneurone populations are selectively impaired, oculomotor neurones are essentially resistant to ALS-related damage. Motoneurone vulnerability has been closely linked to disruptions of calcium signalling. To investigate underlying events, we performed a quantitative analysis of calcium homeostasis in oculomotor neurones from mice by simultaneous patch-clamp recordings in sliced tissue and microfluorometric-calcium measurements. Somatic calcium dynamics were investigated by using a computer-controlled microfluorometric system. In oculomotor neurones, basal calcium concentrations were around 80 nM and depolarisation-induced calcium responses were observed for membrane voltages positive to -40 u1u1u approximately mV1 approximately . Endogenous calcium homeostasis was quantified by using the 'added buffer' approach. The recovery phase of depolarisation-induced calcium transients was well approximated by a mono-exponential function with a decay time constant that showed a linear dependence on dye concentration. The extrapolated time constant in the absence of indicator dye was 1.7 +/- 0.2 s (n = 11 cells, 21C). Endogenous calcium binding ratios (kappa(s)) were found to be 264 +/- 25 (n = 11 cells), indicating that 99.6 % of cytosolic calcium ions were taken up by endogenous buffers. Recovery of calcium transients was characterised by an 'effective' extrusion rate gamma = 156 +/- 20 s-1 (n = 11 cells, 21 C). Endogenous calcium binding ratios in oculomotor neurones were 5- to 6-fold larger compared with those of more vulnerable motoneurones in the nucleus hypoglossus and spinal cord. In a first order approximation, they reduced the volume of local calcium elevations around open calcium channels, lowered peak amplitudes of global calcium transients for a given influx and prolonged calcium recovery times for a given set of uptake and extrusion mechanisms. With respect to motoneurone degeneration, our measurements suggest that the exceptional stability of oculomotor neurones partially results from a specialised calcium homeostasis based on high buffering capacities. Furthermore, they indicate that cellular adaptations that account for rapid calcium signalling in hypoglossal and spinal motoneurones enhance their vulnerability during ALS-related motoneurone disease.