Pivotal Role In Ca2 Research Articles

Identification and Characterization Analysis of Transient Receptor Potential Mucolipin Protein of Laodelphax striatellus Fallén.

Simple SummaryThe small brown planthopper Laodelphax striatellus is a destructive pest of rice, maize and wheat crops in Asia, causing damage by directly sucking phloem sap and transmitting plant viruses, and thus seriously impacting crops yields. Transient receptor potential mucolipin (TRPML) protein plays a vital role in Ca2+ ions release, resulting in membrane trafficking, autophagy and ion homeostasis; however, till now, we have learned little about the TRPML protein of agricultural pests. In this study, we first identified the TRPML of L. striatellus. We analyzed not only the gene evolutionary features and expression profiles but also clarified the protein subcellular localization and lipid-binding characteristics of Ls-TRPML. We found that TRPML is evolutionarily conserved among agricultural pests. Ls-TRPML is predominately expressed in L. striatellus ovary. Moreover, we found that Ls-TRPML localizes in the nuclear membrane in Spodoptera frugiperda cells and the intestine and ovary of L. striatellus. The binding of Ls-TRPML with lipids was detected by lipid-binding assay, indicating the potential role of Ls-TRPML in lipid interaction. Thus, our findings first helped us analyze the gene characterization of Ls-TRPML and then identify the binding of Ls-TRPML with lipids; our findings will broaden our understanding of TRPML’s roles in agricultural pests.Transient receptor potential mucolipin (TRPML) protein in flies plays a pivotal role in Ca2+ ions release, resulting in membrane trafficking, autophagy and ion homeostasis. However, to date, the characterization of TRPML in agricultural pests remains unknown. Here, we firstly reported the TRPML of a destructive pest of gramineous crops, Laodelphax striatellus. The L. striatellus TRPML (Ls-TRPML) has a 1818 bp open reading frame, encoding 605 amino acid. TRPML in agricultural pests is evolutionarily conserved, and the expression of Ls-TRPML is predominately higher in the ovary than in other organs of L. striatellus at the transcript and protein level. The Bac–Bac system showed that Ls-TRPML localized in the plasma membrane, nuclear membrane and nucleus and co-localized with lysosome in Spodoptera frugiperda cells. The immunofluorescence microscopy analysis showed that Ls-TRPML localized in the cytoplasm and around the nuclei of the intestine cells or ovary follicular cells of L. striatellus. The results from the lipid-binding assay revealed that Ls-TRPML strongly bound to phosphatidylinositol-3,5-bisphosphate, as compared with other phosphoinositides. Overall, our results helped is identify and characterize the TRPML protein of L. striatellus, shedding light on the function of TRPML in multiple cellular processes in agricultural pests.

Crosstalk between Renal and Vascular Calcium Signaling: The Link between Nephrolithiasis and Vascular Calcification.

Calcium (Ca2+) is an important mediator of multicellular homeostasis and is involved in several diseases. The interplay among the kidney, bone, intestine, and parathyroid gland in Ca2+ homeostasis is strictly modulated by numerous hormones and signaling pathways. The calcium-sensing receptor (CaSR) is a G protein–coupled receptor, that is expressed in calcitropic tissues such as the parathyroid gland and the kidney, plays a pivotal role in Ca2+ regulation. CaSR is important for renal Ca2+, as a mutation in this receptor leads to hypercalciuria and calcium nephrolithiasis. In addition, CaSR is also widely expressed in the vascular system, including vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) and participates in the process of vascular calcification. Aberrant Ca2+ sensing by the kidney and VSMCs, owing to altered CaSR expression or function, is associated with the formation of nephrolithiasis and vascular calcification. Based on emerging epidemiological evidence, patients with nephrolithiasis have a higher risk of vascular calcification, but the exact mechanism linking the two conditions is unclear. However, a dysregulation in Ca2+ homeostasis and dysfunction in CaSR might be the connection between the two. This review summarizes renal calcium handling and calcium signaling in the vascular system, with a special focus on the link between nephrolithiasis and vascular calcification.

SERCA2a ameliorates cardiomyocyte T-tubule remodeling via the calpain/JPH2 pathway to improve cardiac function in myocardial ischemia/reperfusion mice

Transverse-tubules (T-tubules) play pivotal roles in Ca2+-induced, Ca2+ release and excitation–contraction coupling in cardiomyocytes. The purpose of this study was to uncover mechanisms where sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a) improved cardiac function through T-tubule regulation during myocardial ischemia/reperfusion (I/R). SERCA2a protein expression, cytoplasmic [Ca2+]i, calpain activity, junctophilin-2 (JPH2) protein expression and intracellular localization, cardiomyocyte T-tubules, contractility and calcium transients in single cardiomyocytes and in vivo cardiac functions were all examined after SERCA2a knockout and overexpression, and Calpain inhibitor PD150606 (PD) pretreatment, following myocardial I/R. This comprehensive approach was adopted to clarify SERCA2a mechanisms in improving cardiac function in mice. Calpain was activated during myocardial I/R, and led to the proteolytic cleavage of JPH2. This altered the T-tubule network, the contraction function/calcium transients in cardiomyocytes and in vivo cardiac functions. During myocardial I/R, PD pretreatment upregulated JPH2 expression and restored it to its intracellular location, repaired the T-tubule network, and contraction function/calcium transients of cardiomyocytes and cardiac functions in vivo. SERCA2a suppressed calpain activity via [Ca2+]i, and ameliorated these key indices. Our results suggest that SERCA2a ameliorates cardiomyocyte T-tubule remodeling via the calpain/JPH2 pathway, thereby improving cardiac function in myocardial I/R mice.

Biochemical characterization of four splice variants of mouse Ca2+/calmodulin-dependent protein kinase Iδ.

Ca2+/calmodulin (CaM)-dependent protein kinase Iδ (CaMKIδ) is a Ser/Thr kinase that plays pivotal roles in Ca2+ signalling. CaMKIδ is activated by Ca2+/CaM-binding and phosphorylation at Thr180 by CaMK kinase (CaMKK). In this study, we characterized four splice variants of mouse CaMKIδ (mCaMKIδs: a, b, c and d) found by in silico analysis. Recombinant mCaMKIδs expressed in Escherichia coli were phosphorylated by CaMKK; however, only mCaMKIδ-a and c showed protein kinase activities towards myelin basic protein in vitro, with mCaMKIδ-b and mCaMKIδ-d being inactive. Although mCaMKIδ-a and mCaMKIδ-c underwent autophosphorylation in vitro, only mCaMKIδ-c underwent autophosphorylation in 293T cells. Site-directed mutagenesis showed that the autophosphorylation site is Ser349, which is found in the C-terminal region of only variants c and b (Ser324). Furthermore, phosphorylation of these sites (Ser324 and Ser349) in mCaMKIδ-b and c was more efficiently catalyzed by cAMP-dependent protein kinase in vitro and in cellulo as compared to the autophosphorylation of mCaMKIδ-c. Thus, variants of mCaMKIδ possess distinct properties in terms of kinase activities, autophosphorylation and phosphorylation by another kinase, suggesting that they play physiologically different roles in murine cells.

Contractile Behavior of Mouse Aorta Depends on SERCA2 Isoform Distribution: Effects of Replacing SERCA2a by SERCA2b.

The Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) actively pumps Ca2+ into the sarco/endoplasmic reticulum, thereby regulating intracellular Ca2+ concentrations and associated physiological processes. Different SERCA isoforms have been described (SERCA1, 2, and 3) with SERCA2 playing a pivotal role in Ca2+ homeostasis in cardiovascular tissues. In the heart, SERCA2a is the dominant isoform and has been proposed as therapeutic target in patients with heart failure. In the vasculature, both SERCA2a and SERCA2b are expressed with SERCA2b being the predominant isoform. The physiological role of SERCA2a in the vasculature, however, remains incompletely understood. In the present study, we used gene-modified mice in which the alternative splicing of the SERCA2-encoding gene (Atp2a2), underlying the expression of SERCA2a, is prevented and SERCA2a is replaced by SERCA2b. The resulting SERCA2b/b mice provide a unique opportunity to investigate the specific contribution of SERCA2a versus SERCA2b to vascular physiology. Aortic segments of SERCA2b/b (SERCA2a-deficient) and SERCA2a/b (control) mice were mounted in organ baths to evaluate vascular reactivity. SERCA2b/b aortic rings displayed higher contractions induced by phenylephrine (1 μM). Surprisingly, the initial inositol-3-phosphate mediated phasic contraction showed a faster decay of force in SERCA2b/b mice, while the subsequent tonic contraction was larger in SERCA2b/b segments. Moreover, in the presence of the calcium channel blocker diltiazem (35 μM) SERCA2b/b aortic rings showed higher contractions compared to SERCA2a/b, suggesting that SERCA2a (deficiency) modulates the activity of non-selective cation channels. Additionally, in endothelial cell (EC)-denuded aortic segments, the SERCA-inhibitor cyclopiazonic acid (CPA) caused markedly larger contractions in SERCA2b/b mice, while the increases of cytosolic Ca2+ were similar in both strains. Hence, aortas of SERCA2b/b mice appear to have a stronger coupling of intracellular Ca2+ to contraction, which may be in agreement with the reported difference in intracellular localization of SERCA2a versus SERCA2b. Finally, EC-mediated relaxation by acetylcholine and ATP was assessed. Concentration-response-curves for ATP showed a higher sensitivity of aortic segments of SERCA2b/b mice, while no difference in potency between strains were observed for acetylcholine. In summary, despite the relative low expression of SERCA2a in the murine aorta, our results point toward a distinct role in vascular physiology.

Mn2+ modulates the expression of cellulase genes in Trichoderma reesei Rut-C30 via calcium signaling

BackgroundThe filamentous fungus Trichoderma reesei Rut-C30 is one of the most vital fungi for the production of cellulases, which can be used for biofuel production from lignocellulose. Nevertheless, the mechanism of transmission of external stimuli and signals in modulating cellulase production in T. reesei Rut-C30 remains unclear. Calcium is a known second messenger regulating cellulase gene expression in T. reesei.ResultsIn this study, we found that a biologically relevant extracellular Mn2+ concentration markedly stimulates cellulase production, total protein secretion, and the intracellular Mn2+ concentration of Rut-C30, a cellulase hyper-producing strain of T. reesei. Furthermore, we identified two Mn2+ transport proteins, designated as TPHO84-1 and TPHO84-2, indicating that they are upstream in the signaling pathway that leads to cellulase upregulation. We also found that Mn2+ induced a significant increase in cytosolic Ca2+ concentration, and that this increased cytosolic Ca2+ might be a key step in the Mn2+-mediated regulation of cellulase gene transcription and production. The utilization of LaCl3 to block plasma membrane Ca2+ channels, and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) to interrupt calcium signaling, showed that Mn2+ exerts the induction of cellulase genes via calcium channels and calcium signaling. To substantiate this, we identified a Ca2+/Mn2+ P-type ATPase, TPMR1, which could play a pivotal role in Ca2+/Mn2+ homeostasis and Mn2+ induction of cellulase genes in T. reesei Rut-C30.ConclusionsTaken together, our results revealed for the first time that Mn2+ stimulates cellulase production, and demonstrates that Mn2+ upregulates cellulase genes via calcium channels and calcium signaling. Our research also provides a direction to facilitate enhanced cellulase production by T. reesei.

Low-Intensity Ultrasound Modulates Ca2+ Dynamics in Human Mesenchymal Stem Cells via Connexin 43 Hemichannel.

In recent years, ultrasound has gained attention in new biological applications due to its ability to induce specific biological responses at the cellular level. Although the biophysical mechanisms underlying the interaction between ultrasound and cells are not fully understood, many agree on a pivotal role of Ca2+ signaling through mechanotransduction pathways. Because Ca2+ regulates a vast range of downstream cellular processes, a better understanding of how ultrasound influences Ca2+ signaling could lead to new applications for ultrasound. In this study, we investigated the mechanism of ultrasound-induced Ca2+ mobilization in human mesenchymal stem cells using 47MHz focused ultrasound to stimulate single cells at low intensities (~110mW/cm2). We found that ultrasound exposure triggers opening of connexin 43 hemichannels on the plasma membrane, causing release of ATP into the extracellular space. That ATP then binds to G-protein-coupled P2Y1 purinergic receptors on the membrane, in turn activating phospholipase C, which evokes production of inositol trisphosphate and release of Ca2+ from intracellular stores.

Calcium as a Trojan horse in mental diseases-The role of PMCA and PMCA-interacting proteins in bipolar disorder and schizophrenia.

Although first mentions about calcium disturbances in psychiatric diseases appeared more than 30 years ago, the most recent genomic and proteomic findings confirmed a significant role of Ca2+ and Ca2+-regulated pathways in development of neuropathological processes, including bipolar disorder and schizophrenia. Moreover, last decades have shown that due to multifactorial nature of both diseases, impairment in neuronal calcium homeostasis may depend not only on disturbed Ca2+ entry system, but also on altered extrusion system. A pivotal role in Ca2+ clearance mechanism is played by plasma membrane Ca2+-ATPase (PMCA), the enzyme responsible for returning the elevated levels of cytosolic Ca2+ back to the resting state. In this paper we summarize the current knowledge about the role of PMCA in bipolar disorder and schizophrenia pathologies, as well as the contribution of several proteins that by interaction with PMCA modify signal transduction mechanisms.

(Nanocarbons Division Richard E. Smalley Research Award Address) Artificial Photosynthesis Utilizing Nanocarbons

Sustainable and clean energy resources using solar energy alternative to fossil fuels are urgently required in order to stop global warming and solve global energy and environmental issues. This award lecture focuses on our long-term developments in artificial photosynthesis for solar fuel production and its usage by utilizing nanocarbon materials. We have developed a number of photosynthetic reaction center models composed of metalloporphyrins and fullerenes with comparable charge-separated state lifetimes with that of the natural photosynthetic reaction center.[1-3] The light-harvesting unit and the charge-separation unit have been combined by utilizing coordination bonds, hydrogen bond, electrostatic interactions and p-p interactions.[4,5] The efficient photosynthetic reaction center models have been successfully combined with the catalytic water reduction unit to attain the most efficient photocatalytic hydrogen evolution system.[6-8] The activation of hydrogen has also been achieved by developing the first structural and functional model of hydrogenases,[9] which enables to replace precious Pt catalyst by much more earth-abundant metal catalyst for hydrogen evolution.[10] With regard to water oxidation, we have developed efficient and robust water oxidation catalysts using earth-abundant metal oxides nanoparticles.[11] We have also clarified the significant role of redox-inactive metal ions on enhancement of electron-transfer reactions of high-valent metal-oxo complexes, which is classified as metal ion-coupled electron transfer,[12,13] in relation with the pivotal role of Ca2+ in the oxygen evolving complex (OEC) in Photosystem II.[14,15] By combination of the catalytic four-electron water oxidation and the photocatalytic two-electron reduction of O2 using solar energy, we have succeeded in production of hydrogen peroxide (H2O2) as a solar fuel from water and O2 in the air.[16,17] We have also achieved efficient photocatalytic production of H2O2 from the most earth abundant seawater instead of precious pure water and O2 in the air using a two-compartment photoelectro-chemical cell with WO3 as a photocatalyst for water oxidation and a cobalt chlorin complex supported on a glassy-carbon substrate for the selective two-electron reduction of O2.[18] The highest solar energy conversion efficiency was determined to be 6.6% under simulated solar illumination adjusted to 0.05 sun after 1 h photocatalytic reaction (0.89% under 1sun illumination) when WO3 was replaced by the surface modified BiVO4 with iron(III) oxide(hydroxide) (FeO(OH)) as a water oxidation catalyst in the photoanode.[20] We have also developed one-compartment H2O2 fuel cells using H2O2 produced in seawater as a solar fuel and earth-abundant metal catalysts.[18,20,22] Thus, the combination of the photocatalytic H2O2 production from seawater and O2 using solar energy with one-compartment H2O2 fuel cells provides on-site production and usage of H2O2 as a more useful and promising solar fuel than H2.[18] References [1] Fukuzumi, S.; Ohkubo, K.; Suenobu, T. Acc. Chem. Res. 2014, 47, 1455-1464. [2] Fukuzumi, S. Phys. Chem. Chem. Phys. 2008, 10, 2283. [3] Fukuzumi, S.; Ohkubo, K. J. Mater. Chem. 2012, 22, 4575. [4] Fukuzumi, S.; Ohkubo, K.; D’Souza, F.; Sessler, J. L. Chem. Commun. 2012, 48, 9801-9815. [5] Yamada, M.; Ohkubo, K.; Shionoya, M.; Fukuzumi, S. J. Am. Chem. Soc. 2014, 136, 13240-13248. [6] Fukuzumi, S.; Yamada, Y.; Suenobu, T.; Ohkubo, K.; Kotani, H, Energy Environ. Sci. 2011, 4, 2754-2766. [7] Fukuzumi, S.; Yamada, Y. J. Mater. Chem. 2012, 22, 24284-24296. [8] Fukuzumi, S. Curr. Opinion Chem. Biol. 2015, 25, 18-26. [9] Ogo, S.; Kabe, R.; Uehara, K.; Kure, B.; Nishimura, T.; Menon, S. C.; Harada, R.; Fukuzumi, S.; Higuchi, Y.; Ohhara, T.; Tamada, T.; Kuroki, R. Science 2007, 316, 585-587. [10] Yamada, Y.; Miyahigashi, T.; Kotani, H.; Ohkubo, K.; Fukuzumi, S. Energy Environ. Sci. 2012, 5, 6111-6118. [11] Hong, D.; Yamada, Y.; Nagatomi, T.; Takai, Y.; Fukuzumi, S. J. Am. Chem. Soc. 2012, 134, 19572-19575. [12] Fukuzumi, S. Prog. Inorg. Chem. 2009, 56, 49-153. [13] Fukuzumi, S.; Ohkubo, K.; Lee, Y.-M.; Nam, W. Chem.–Eur. J. 2015, 21, 17548-17559. [14] Fukuzumi, S.; Morimoto, Y.; Kotani, H.; Naumov, P.; Lee, Y.-M.; Nam, W. Nat. Chem. 2010, 2, 756-759. [15] Bang, S.; Lee, Y.-M.; Hong, S.; Nishida, Y.; Seo, M. S.; Sarangi, R.; Fukuzumi, S.; Nam, W. Nat. Chem. 2014, 6, 934-940. [16] Kato, S.; Jung, J.; Suenobu, T.; Fukuzumi, S. Energy Environ. Sci. 2013, 6, 3756-3764. [17] Isaka, Y.; Kato, S.; Hong, D.; Suenobu, T.; Yamada, Y.; Fukuzumi, S. J. Mater. Chem. A 2015, 3, 12404-12412. [18] Mase, K.; Yoneda, M.; Yamada, Y.; Fukuzumi, S. Nat. Commun. 2016, 7, 11470. [19] Mase, K.; Yoneda, M.; Yamada, Y.; Fukuzumi, S. ACS Energy Lett. 2016, 1, 913-919. [20] Yamada, Y.; Yoneda, M.; Fukuzumi, S. Energy Environ. Sci. 2015, 8, 1698-1701. [21] Fukuzumi, S.; Yamada, Y. ChemElectroChem 2016, 3, in press.

The Pivotal Role of Ca2+ Homeostasis in PBDE-47-Induced Neuronal Apoptosis.

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants and are ubiquitous in the environment and human tissues. Recent evidence has demonstrated that PBDE-induced neurotoxicity is associated with neuronal apoptosis via interfering with the calcium ion (Ca2+) homeostasis; however, the underlying mechanisms remain elusive. Thus, we sought to investigate the role of Ca2+ homeostasis in PBDE-47-induced neuronal apoptosis. Here, we showed that PBDE-47 significantly decreased neuronal number while increased neuronal apoptosis in vitro and in vivo, as manifested by an increased percentage of Annexin V-positive staining cells and caspase-3 activation in human neuroblastoma SH-SY5Y cells and hippocampal neurons of rats. Further study identified that PBDE-47 elicited ΔΨm collapse following an early and sustained [Ca2+] i, overload, as well as stimulated cytochrome c release from mitochondria into the cytosol in SH-SY5Y cells and rat hippocampal tissue. Interestingly, the extracellular Ca2+ chelator ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) blocked PBDE-47-induced [Ca2+] i elevation, ΔΨm collapse, cytochrome c release, and caspase-3 activation in SH-SY5Y cells, whereas the intracellular Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) had no influences on them, indicating that the [Ca2+] i overload originates primarily from extracellular Ca2+ component rather than from intracellular calcium storage and that the increase in [Ca2+] i is a major contributor to ΔΨm collapse and subsequent neuronal apoptosis. Overall, these findings suggest that PBDE-47 affects Ca2+ homeostasis as a crucial event in activation of neuronal death associated with mitochondria and provide novel insight into the mechanism of action underlying PBDE neurotoxicity.

The Phosphatase-Resistant Isoform of CaMKI, Ca2+/Calmodulin-Dependent Protein Kinase Iδ (CaMKIδ), Remains in Its “Primed” Form without Ca2+ Stimulation

Ca²⁺/calmodulin-dependent protein kinase I (CaMKI) is known to play pivotal roles in Ca²⁺ signaling pathways. Four isoforms of CaMKI (α, β, γ, and δ) have been reported so far. CaMKI is activated through phosphorylation by the upstream kinase, CaMK kinase (CaMKK), and phosphorylates downstream targets. When CaMKI was transiently expressed in 293T cells, CaMKIα was not phosphorylated at all under low-Ca²⁺ conditions in the cells. In contrast, we found that CaMKIδ was significantly phosphorylated and activated to phosphorylate cAMP response element-binding protein (CREB) under the same conditions. Herein, we report that the sustained activation of CaMKIδ is ascribed to its phosphatase resistance resulting from the structure of its N-terminal region. First, we examined whether CaMKIδ is more readily phosphorylated by CaMKK than CaMKIα, but no significant difference was observed. Next, to compare the phosphatase resistance between CaMKIα and CaMKIδ, we assessed the dephosphorylation of the phosphorylated CaMKIs by CaMK phosphatase (CaMKP/PPM1F). Surprisingly, CaMKIδ was hardly dephosphorylated by CaMKP, whereas CaMKIα was significantly dephosphorylated under the same conditions. To date, there have been no detailed reports concerning dephosphorylation of CaMKI. Through extensive analysis of CaMKP-catalyzed dephosphorylation of various chimeric and point mutants of CaMKIδ and CaMKIα, we identified the amino acid residues responsible for the phosphatase resistance of CaMKIδ (Pro-57, Lys-62, Ser-66, Ile-68, and Arg-76). These results also indicate that the phosphatase resistance of CaMKI is largely affected by only several amino acids in its N-terminal region. The phosphatase-resistant CaMKI isoform may play a physiological role under low-Ca²⁺ conditions in the cells.

Interactions between sarco-endoplasmic reticulum and mitochondria in cardiac and skeletal muscle – pivotal roles in Ca2+ and reactive oxygen species signaling

Mitochondria are strategically and dynamically positioned in the cell to spatially coordinate ATP production with energy needs and to allow the local exchange of material with other organelles. Interactions of mitochondria with the sarco-endoplasmic reticulum (SR/ER) have been receiving much attention owing to emerging evidence on the role these sites have in cell signaling, dynamics and biosynthetic pathways. One of the most important physiological and pathophysiological paradigms for SR/ER-mitochondria interactions is in cardiac and skeletal muscle. The contractile activity of these tissues has to be matched by mitochondrial ATP generation that is achieved, at least in part, by propagation of Ca(2+) signals from SR to mitochondria. However, the muscle has a highly ordered structure, providing only limited opportunity for mitochondrial dynamics and interorganellar interactions. This Commentary focuses on the latest advances in the structure, function and disease relevance of the communication between SR/ER and mitochondria in muscle. In particular, we discuss the recent demonstration of SR/ER-mitochondria tethers that are formed by multiple proteins, and local Ca(2+) transfer between SR/ER and mitochondria.

821 AGE-RELATED DIFFERENCES IN BLOOD PRESSURE RESPONSE TO ACUTE BLOCKADE OF CALCIUM INFLUX AND CALCIUM SENSITIZATION IN WISTAR-KYOTO AND SPONTANEOUSLY HYPERTENSIVE RATS

Background: Spontaneously hypertensive rats (SHR) are characterized by augmented Ca2+ influx through L-type voltage-dependent calcium channels (L-VDCC), which is one of the most important factors responsible for increased blood pressure (BP). The altered BP regulation in hypertension also includes RhoA/Rho-kinase pathway playing pivotal role in Ca2+ sensitization. The ontogenetic development of Ca2+ influx and Ca2+ sensitization has not been fully examined. Our study was aimed to determine the contribution of these two mechanisms to BP maintenance in conscious normotensive Wistar-Kyoto rats (WKY) and SHR at different ages. Design and methods: BP response to acute intravenous administration of cumulative doses of L-VDCC blocker (nifedipine) or Rho kinase inhibitor (fasudil) was determined in 3-, 5-, 7-, 13-, and 26-week-old WKY and SHR. Results: There were no significant differences in BP response to nifedipine induced L-VDCC blockade in all age groups of WKY rats, while in SHR BP response increased with age and/or hypertension level. The same was true for Rho kinase inhibition. In 3-week-old rats nifedipine-sensitive BP components were identical in both strains, whereas BP response to fasudil was greater in 3-week-old WKY than in prehypertensive SHR. Finally, the fasudil-sensitive BP component was greater in 13- and 26-week-old SHR than WKY. Conclusions: Ca2+ influx through L-VDCC and Ca2+ sensitization mediated by RhoA/Rho-kinase pathway are involved in the regulation of vascular tone in both strains. Both mechanisms are augmented in rats with developed hypertension, but RhoA/Rho-kinase pathway seems to be diminished in prehypertensive SHR. Partially supported by grant 304/12/0259 (GA CR).

Nuclear Sphingolipid Metabolism

Nuclear lipid metabolism is implicated in various processes, including transcription, splicing, and DNA repair. Sphingolipids play roles in numerous cellular functions, and an emerging body of literature has identified roles for these lipid mediators in distinct nuclear processes. Different sphingolipid species are localized in various subnuclear domains, including chromatin, the nuclear matrix, and the nuclear envelope, where sphingolipids exert specific regulatory and structural functions. Sphingomyelin, the most abundant nuclear sphingolipid, plays both structural and regulatory roles in chromatin assembly and dynamics in addition to being an integral component of the nuclear matrix. Sphingosine-1-phosphate modulates histone acetylation, sphingosine is a ligand for steroidogenic factor 1, and nuclear accumulation of ceramide has been implicated in apoptosis. Finally, nuclear membrane-associated ganglioside GM1 plays a pivotal role in Ca(2+) homeostasis. This review highlights research on the factors that control nuclear sphingolipid metabolism and summarizes the roles of these lipids in various nuclear processes.

Temperature effects on Ca2+ cycling in scombrid cardiomyocytes: a phylogenetic comparison

Specialisations in excitation-contraction coupling may have played an important role in the evolution of endothermy and high cardiac performance in scombrid fishes. We examined aspects of Ca(2+) handling in cardiomyocytes from Pacific bonito (Sarda chiliensis), Pacific mackerel (Scomber japonicus), yellowfin tuna (Thunnus albacares) and Pacific bluefin tuna (Thunnus orientalis). The whole-cell voltage-clamp technique was used to measure the temperature sensitivity of the L-type Ca(2+) channel current (I(Ca)), density, and steady-state and maximal sarcoplasmic reticulum (SR) Ca(2+) content (ssSR(load) and maxSR(load)). Current-voltage relations, peak I(Ca) density and charge density of I(Ca) were greatest in mackerel and yellowfin at all temperatures tested. I(Ca) density and kinetics were temperature sensitive in all species studied, and the magnitude of this response was not related to the thermal preference of the species. SR(load) was greater in atrial than in ventricular myocytes in the Pacific bluefin tuna, and in species that are more cold tolerant (bluefin tuna and mackerel). I(Ca) and SR(load) were particularly small in bonito, suggesting the Na(+)/Ca(2+) exchanger plays a more pivotal role in Ca(2+) entry into cardiomyocytes of this species. Our comparative approach reveals that the SR of cold-tolerant scombrid fishes has a greater capacity for Ca(2+) storage. This specialisation may contribute to the temperature tolerance and thermal niche expansion of the bluefin tuna and mackerel.

From antibodies to adiponectin: role of ERp44 in sizing and timing protein secretion

A large fraction of the proteome is synthesized and folded in the endoplasmic reticulum (ER), a multifunctional compartment also playing pivotal roles in Ca(2+) storage, redox homeostasis and signalling. From the ER, secretory proteins begin their journey towards their final destinations, the organelles of the exocytic and endocytic compartments, the plasma membrane or the extracellular space. Fidelity of protein-based intracellular communication is guaranteed by quality control (QC) mechanisms located at the ER-Golgi interface, which restrict forward transport to native proteins. QC is used also to time or shape the secretome. Furthermore, professional secretory cells face a problem of quantity, as well as quality of their protein products. This essay summarizes recent findings that identify ERp44 as a key regulator of protein secretion, Ca(2+) signalling and redox regulation.

Molecular cloning and expression profiles of calreticulin gene from the diamondback moth, Plutella xylostella

AbstractCalreticulin (CRT) plays pivotal roles in Ca2+ homeostasis, molecular chaperoning, infection, inflammation and innate immunity. In an attempt to study the involvement of CRT in innate immunity, the full‐length cDNA of calreticulin (PxCRT) was cloned from the diamondback moth, Plutella xylostella. It consists of 1674 bp (excluding poly‐A tail) with a longest open reading frame (ORF) of 1197 bp encoding 398 amino acids. In silico analysis of PxCRT ORF reveals that it has various repeat motifs and endoplasmic reticulum retention signal found in all the calreticulin proteins. As expected, high amino acid sequence identities were found from other CRTs identified from Bombyx mori (87%), Galleria mellonella (87%), Apis mellifera (74%), Anopheles gambiae (74%), Tribolium castaneum (73%), Culex quinquefasciatus (73%), Rhodnius prolixus (72%), Nasonia vitripennis (71%), Drosophila melanogaster (71%) and Haemaphysalis qinghaiensis (68%). During development, P. xylostella expressed PxCRT predominantly in the pupal stage. In addition, spatial expression pattern analysis indicates that PxCRT was highly expressed in the silk gland. PxCRT mRNA, furthermore, was strongly induced 3 to 6 h after laminarin treatment, suggesting that PxCRT appears to be involved in immune responses and also plays an important role in the silk gland.

Characterization of Ca2+ Signalling in Postnatal Mouse Retinal Ganglion Cells: Involvement of OPA1 in Ca2+ Clearance

Purpose: The regulation of Ca2+ entry and removal is a fine-tuned process which remains not well understood in mouse retinal ganglion cells (RGCs). The latter are known to be sensitive to dysfunctions of mitochondria, organelles playing a pivotal role in Ca2+ reuptake.Methods: We first described the Ca2+ signals of RGCs in response to varied drugs with Fura-2 imaging, and secondly tested the role of optic atrophy 1 or OPA1, the gene responsible for Autosomal Dominant Optic Atrophy, on mitochondrial ability to capture intracellular Ca2+ in cells transfected with the OPA1 small interfering ribonucleic acids (siRNAs).Results: In control RGCs, K+-evoked [Ca2+]i increase was blocked by the Ca2+ channel antagonists (Ni2++ Cd2+) and GABAA receptor agonist muscimol-induced [Ca2+]i responses were attenuated by the GABAA receptor antagonists, picrotoxin and gabazine. We also prove the presence of NMDA and AMPA/Kainate (glutamate receptor agonists) responsive receptors in this model. Application of cyclopiazonic acid, an inhibitor of Ca2+-ATPase pumps of the intracellular Ca2+ stores, induced an increase in [Ca2+]i while ryanodine or caffeine had no effect on resting [Ca2+]i. Spontaneous Ca2+ oscillations in contacting neurons highlighted the importance of cross-talks between RGCs during maturation. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), induced robust raises of intracellular Ca2+ after K+ application, with a more pronounced effect in cells silenced for OPA1, which could lead to cell death.Conclusions: Our results indicate an important role of OPA1 in mitochondrial dependent Ca2+ homeostasis and cell survival in RGCs, suggesting a possible patho-physiological mechanism involved in inherited optic neuropathies.

Combined Proteomic and Cytological Analysis of Ca2+-Calmodulin Regulation inPicea meyeriPollen Tube Growth

Ca2+-calmodulin (Ca2+-CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca2+-CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca2+-CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca2+-CaM signaling rapidly induced an increase in extracellular Ca2+ influx, resulting in dramatically increased cytosolic Ca2+ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca2+-CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca2+-CaM in the regulation of tip growth machinery.

Ca2+/Calmodulin-dependent Protein Kinase Enriched in Cerebellar Granule Cells: Identification of a novel neuronal calmodulin-dependent protein kinase

Ca2+-sensitive protein kinases are thought to play a pivotal role in Ca2+-mediated neuronal communication. We describe here the cloning, purification, and characterization of a major Ca2+/calmodulin-dependent, brain-specific protein kinase which is particularly enriched in cerebellar granule cells. The enzyme is comprised of Mr 65,000 and 67,000 polypeptides which copurify to homogeneity and phosphorylate synapsin I. The protein kinase is coded for by two poly(A+) RNAs of 2.0 and 3.5 kilobases which probably derive from a single gene. Two cDNA inserts, one of 198 base pairs and one of 1225 base pairs, contain a total of 677 base pairs of the protein coding sequence which includes sequences homologous to other calmodulin-dependent protein kinases including part of the calmodulin-binding domain. The surprising presence of extended sequences which are enriched in glutamate residues may influence the subcellular distribution of this kinase. Immunohistochemical localization with an affinity-purified antibody reveals that whereas the enzyme is expressed in several neuronal subpopulations, it is exceptionally enriched in the granule cells of the cerebellum. The relevance of the biochemical, molecular, and histologic properties of this enzyme is discussed in the context of neuronal Ca2+ signaling.