An electron microscopic—cytochemical localization of plasma membrane Ca^2＋—ATPase activity in poplar apical bud cells during the induction of dormancy by short—day photoperiods

Plasma membrane(PM) Ca^2 -ATPase activity in poplar apical bud meristematic cells during short-day(SD)-induced dormancy development was examined by a cerium precipitation EM-cytochemical method.Ca^2 -ATPase activity,indicated by the status of cerium phosphate precipitated grains,was localized mainly on the interior face(cytoplasmic side) of the PM when plants were grown under long days and reached a deep dormancy.A few reaction products were also observed on the nuclear envelope.When plant buds were developing dormancy after 28 to 42 d of SD exposure,almost no reaction products were present on the interior face of the PM.In contrast,a large number of cerium phosphate precipitated grains were distributed on the exterior face of the PM.After 70 d of SD exposure,when buds had developed a deep dormancy,the reaction products of Ca^2 -ATPase activity again appeared on the interior face of the PM.The results seemed suggesting that two kinds of Ca^2 -ATP ases may be present on the PM during the SD-induced dormancy in poplar.One is the Ca^2 -pumping ATPase,which is located on the interior face of the PM,for maintaining and restoring the Ca^2 homeostasis.The other might be and ecto-Ca^2 -ATPase,which is located on the exterior face of the PM,for the exocytosis of cell wall materials as suggested by the fact of the cell wall thickening during the dormancy development in poplar.     

Ethanol breaks dormancy of the potato tuber apical bud

Growing potato tubers or freshly harvested mature tubers have a dormant apical bud. Normally, this dormancy is spontaneously broken after a period of maturation of the tuber, resulting in the growth of a new sprout. Here it is shown that in in vitro-cultured growing and maturing tubers, ethanol can rapidly break this dormancy and re-induce growth of the apical bud. The in vivo promoter activity of selected genes during this secondary growth of the apical bud was monitored, using luciferase as a reporter. In response to ethanol, the expression of carbohydrate-storage, protein-storage, and cell division-related genes are rapidly down-regulated in tuber tissue. It was shown that dormancy was broken by primary but not by secondary alcohols, and the effect of ethanol on sprouting and gene expression in tuber tissue was blocked by an inhibitor of alcohol dehydrogenase. By contrast, products derived from alcohol dehydrogenase activity (acetaldehyde and acetic acid) did not induce sprouting, nor did they affect luciferase reporter gene activity in the tuber tissue. Application of an inhibitor of gibberellin biosynthesis had no effect on ethanol-induced sprouting. It is suggested that ethanol-induced sprouting may be related to an alcohol dehydrogenase-mediated increase in the catabolic redox charge [NADH/(NADH+NAD+)].     

淹水玉米幼苗根尖分生细胞内Ca2+超微细胞化学定位

采用焦锑酸钾沉淀法,对遭受淹水胁迫的玉米幼苗初生根根尖分生细胞内钙离子分布变化情况进行了电镜细胞化学观察。在正常状态下,根尖分生细胞内Ca^2＋沉淀颗粒的分布较少．主要位于细胞核和细胞质中。在淹水1h后,根尖分生细胞内呈现有大量Ca^2＋沉淀颗粒分布,细胞核和细胞质中分布的Ca^2＋沉淀颗粒密度,远大于正常细胞。随着淹水时间的延长,根尖分生细胞的细胞核和细胞质中分布的Ca^2＋沉淀颗粒呈现不断增多的趋势,而液泡中分布的Ca^2＋沉淀颗粒则逐步明显减少。根据实验结果本文对受淹根尖分生细胞的死亡与Ca^2＋分布变化的关系进行了研究。     

Alterations in Ca2+-channels during the development of diabetic cardiomyopathy

In order to examine the status of Ca²⁺ channels in heart sarcolemma during the development of diabetes, rats were injected intravenously with 65 mg/kg streptozotocin and hearts were removed 1, 3 and 8 weeks later. Crude membranes from the ventricular muscle were prepared and the specific binding of ³H-nitrendipine was studied by employing different concentrations of this Ca ²⁺-antagonist. A significant decrease in both dissociation constant and maximal number of 3H-nitrendipine binding was observed in 3 and 8 weeks diabetic preparations. No such alterations were evident in diabetic brain membranes. Treatment of diabetic animals with insulin prevented the occurrence of these changes in the myocardium. The altered ³H-nitrendipine binding characteristics in diabetic heart membranes may not be due to the high levels of circulating catecholamines in this experimental model because no such changes were seen upon injecting a high dose (40 mg/kg) of isoproterenol in rats for 24 hr. The reduced number of ³H-nitrendipine binding sites may decrease Ca²⁺-influx through voltage sensitive Ca²⁺ channels and partly explain the depressed cardiac contractile force development in chronic diabetes whereas the increased affinity of Ca²⁺ channels may partly explain the increased sensitivity of diabetic heart to Ca ²⁺.     

Structure, function and subcellular localization of a human platelet Ca2+-ATPase

Human platelets contain a Ca2+-ATPase in internal membranes that is essential for Ca2+ homeostasis. This Ca2+ pump has enzymatic properties quite similar to the sarcoplasmic reticulum (SR) Ca2+ pumps. Antibodies against the SR Ca2+ pump crossreact with the human platelet protein. However, the platelet Ca2+-ATPase is approximately 10 kD larger than the SR pumps and exhibits a larger mRNA coding for the protein in a megakaryocyte tumor cell line. In addition, the platelet Ca2+-pump may be localized in specialized internal membrane structures that function in Ca2+ uptake and release. These results suggest that the platelet Ca2+-ATPase may represent a new class of internal membrane Ca2+-pumps.     

Alterations in cardiac membrane Ca2+ transport during oxidative stress

Although cardiac dysfunction due to ischemia-reperfusion injury is considered to involve oxygen free radicals, the exact manner by which this oxidative stress affects the myocardium is not clear. As the occurrence of intracellular Ca²⁺ overload has been shown to play a critical role in the genesis of cellular damage due to ischemia-reperfusion, this study was undertaken to examine whether oxygen free radicals are involved in altering the sarcolemmal Ca²⁺-transport activities due to reperfusion injury. When isolated rat hearts were made globally ischemic for 30 min and then reperfused for 5 min, the Ca²⁺ -pump and Na⁺-Ca²⁺ exchange activities were depressed in the purified sarcolemmal fraction; these alterations were prevented when a free radical scavenger enzymes (superoxide dismutase plus catalase) were added to the reperfusion medium. Both the Ca²⁺- pump and Na⁺- Ca²⁺ exchange activities in control heart sarcolemmal preparations were depressed by activated oxygen-generating systems containing xanthine plus xanthine oxidase and H₂O₂; these changes were prevented by the inclusion of superoxide dismutase and catalase in the incubation medium. These results support the view that oxidative stress during ischemia-reperfusion may contribute towards the occurrence of intracellular Ca²⁺ overload and subsequent cell damage by depressing the sarcolemmal mechanisms governing the efflux of Ca²⁺ from the cardiac cell.     

Spontaneous Ca^2＋ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca^2＋ pools

INTRODUCTION In vascular smooth muscle, as in other types of muscle,an increase in intracellular Ca2 is the immediate triggerfor contraction, which ultimately determines vascular toneand peripheral resistance. In the past 12 years, investiga-tors have …     

Profilin is required for Ca2+ homeostasis and Ca2+-modulated bud formation in yeast

A cls5-1 mutant of Saccharomyces cerevisiae is specifically sensitive to high concentrations of Ca²⁺, with elevated intracellular calcium content and altered cell morphology in the presence of 100 mM Ca²⁺. To reveal the mechanisms of the Ca²⁺-sensitive phenotype, we investigated the gene responsible and its interacting network. We demonstrated that CLS5 is identical to PFY1, encoding profilin. Involvement of profilin in the maintenance of intracellular Ca²⁺ homeostasis was supported by the fact that both exchangeable and non-exchangeable intracellular Ca²⁺ pools in the cls5-1 mutant are higher than those of the wild-type strain. Several mutations of the genes whose proteins physically interact with profilin resulted in the Ca²⁺-sensitive phenotype. Examination of the intracellular Ca²⁺ pools indicated that Bni1p, Bem1p, Rho1p, and Cla4p are also required for the maintenance of Ca²⁺ homeostasis. Quantitative morphological analysis revealed that the Ca²⁺-induced morphological changes in cls5-1 cells are similar to bem1 and cls4-1 cells. Common Ca²⁺-induced morphological changes were an increase in cell size and a decrease of the ratio of budded cells in the population. Since a mutation allele of cls4-1 is located in the CDC24 gene, we suggest that profilin, Bem1p, and Cdc24p are required for Ca²⁺-modulated bud formation. Thus, profilin is involved in Ca²⁺ regulation in two ways: the first is Ca²⁺ homeostasis by coordination with Bni1p, Bem1p, Rho1p, and Cla4p, and the second is the requirement of Ca²⁺ for bud formation by coordination with Bem1p and Cdc24p.     

Alterations in subcellular localization of p38 MAPK potentiates endothelin-stimulated COX-2 expression in glomerular mesangial cells

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide with mitogenic actions linked to activation of tyrosine kinase signaling pathways. ET-1 induces cyclooxygenase-2 (COX-2), an enzyme that converts arachidonic acid to pro-inflammatory eicosanoids. Activation of each of the three major mitogen-activated protein kinase (MAPK) pathways, ERK1/2, JNK/SAPK, and p38 MAPK (p38), have been shown to enhance the expression of COX-2. Negative regulation of MAPK may occur via a family of dual specificity phosphatases referred to as mitogen-activated protein kinase phosphatases (MKP). The goal of this work was to test the hypothesis that wild type MKP-1 regulates the expression of ET-1-induced COX-2 expression by inhibiting the activation of p38 in cultured glomerular mesangial cells (GMC). An adenovirus expressing both wild type and a catalytically inactive mutant of MKP-1 (MKP-1/CS) were constructed to study ET-1-regulated MAPK signaling and COX-2 expression in cultured GMC. ET-1 stimulated the phosphorylation of ERK and p38 alpha MAPK and induced the expression of COX-2. Expression of COX-2 was partially blocked by U0126, a MEK inhibitor, and SB 203580, a p38 MAPK inhibitor. Adenoviral expression of MKP-1/CS augmented basal and ET-1-induced phosphorylation of p38 alpha MAPK with less pronounced effects on ERK1/2 phosphorylation. Ectopic expression of wild type MKP-1 blocked the phosphorylation of p38 alpha MAPK by ET-1 but increased the phosphorylation of p38 gamma MAPK. Co-precipitation studies demonstrated association of MKP-1 with p38 alpha MAPK and ERK1/2. Immunofluorescent image analysis demonstrated trapping of phospho-p38 MAPK in the cytoplasm by MKP-1/CS/green fluorescent protein. ET-1-stimulated expression of COX-2 was increased in MKP-1/CS versus LacZ or green fluorescent protein-infected control cells. These results indicate that MKP-1 demonstrates a relative selectivity for p38 alpha MAPK versus p38 gamma MAPK in GMC and is likely to indirectly regulate the expression of COX-2.     

Expression of beta-tubulin during dormancy induction and release in apical and axillary buds of five woody species

Cell cycle activity was studied in apical and axillary buds of Norway maple ( Acer platanoides L.), apple ( Malus ' M9 ') , pedunculate oak ( Quercus robur L.), Scots pine ( Pinus sylvestris L.) and rose ( Rosa corymbifera 'Laxa') during dormancy induction and release. Flow cytometric analyses revealed that in dormant buds, cells mainly were quiescent at the G₀/G₁ phase, while in non-dormant buds, a significantly higher frequency of G₂ cells was found in all species. In western blots accumulation of 55 kDa beta -tubulin was found in active growing plant material, whereas in dormant buds the accumulation was much lower or below detection level. It was observed for all species that during dormancy induction the amount of beta -tubulin decreased, while during dormancy release a fast accumulation of beta -tubulin occurred. The dynamics of the beta -tubulin accumulation reflected the dormancy status of tree buds of the five species studied suggesting that the beta -tubulin level might be useful as a marker for the dormancy status in buds of temperate woody species.     

The interaction of microgravity and ethylene on the ultrastructure cell and Ca2+ localization in soybean hook hypocotyl

Calcium ions are secondary messenger in numerous cellular processes of plant grown at 1 g. Ca2+ are connected with oxygen atoms, of pectin carboxy groups and/or with H(+)-groups of protein (Roux and Slocum, 1982; Hepler and Wayne, 1985). The influence of altered gravity on the calcium balance in some cells is established. The increased synthesis of ethylene in plant grown in microgravity caused the change of the structural-functional organization of cell (Hensel and Iversen, 1980; Hilaire et al., 1996). Available data put the new question: how do high ethylene level and microgravity influence on the redistribution of Ca2+ in cell of seedling in early stage of growth? Therefore, the goal of our data was the comparable study of the cell ulltrastructure and localization of Ca2+ in hook hypocotyl of soybean seedling under interaction of microgravity and ethylene.     

Apical localization of a functional TRPC3/TRPC6-Ca2+-signaling complex in polarized epithelial cells. Role in apical Ca2+ influx

Receptor-coupled [Ca2+]i increase is initiated in the apical region of epithelial cells and has been associated with apically localized Ca2+-signaling proteins. However, localization of Ca2+ channels that are regulated by such Ca2+-signaling events has not yet been established. This study examines the localization of TRPC channels in polarized epithelial cells and demonstrates a role for TRPC3 in apical Ca2+ uptake. Endogenously and exogenously expressed TRPC3 was localized apically in polarized Madin-Darby canine kidney cells (MDCK) and salivary gland epithelial cells. In contrast, TRPC1 was localized basolaterally, whereas TRPC6 was detected in both locations. Localization of Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and phospholipase Cbeta1 and -beta2 was also predominantly apical. TRPC3 co-immunoprecipitated with endogenous TRPC6, phospholipase Cbetas, Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and syntaxin 3 but not with TRPC1. Furthermore, 1-oleoyl-2-acetyl-sn-glycerol (OAG)-stimulated apical 45Ca2+ uptake was higher in TRPC3-MDCK cells compared with control (MDCK) cells. Bradykinin-stimulated apical 45Ca2+ uptake and transepithelial 45Ca2+ flux were also higher in TRPC3-expressing cells. Consistent with this, OAG induced [Ca2+]i increase in the apical, but not basal, region of TRPC3-MDCK cells that was blocked by EGTA addition to the apical medium. Most importantly, (i) TRPC3 was detected in the apical region of rat submandibular gland ducts, whereas TRPC6 was present in apical as well as basolateral regions of ducts and acini; and (ii) OAG stimulated Ca2+ influx into dispersed ductal cells. These data demonstrate functional localization of TRPC3/TRPC6 channels in the apical region of polarized epithelial cells. In salivary gland ducts this could contribute to the regulation of salivary [Ca2+] and secretion.     

Ultracytochemical localization of Ca2+ during the phloem ganglion development in Phyllostachys edulis

Ultracytochemical localization of Ca2+ was investigated using the potassium pyroantimonate precipitation method during the development of phloem ganglion.The result showed that Ca2+ was mainly localized in the cell wall and intercellular spaces in the initiating phase.With the development of the phloem ganglion,the distribution of Ca2+ transferred to the vacuole,and the Ca2+ deposits in the cell wall and intercellular space decreased.At the later stage of the developmental phase.Ca2+ was distributed in the tonoplast and vacuole phagocytosis,and the vacuole became the main calcium storage in this phase.At the early stage of maturation of the phloem ganglion,most of the phloem ganglion cells'vacuoles cracked,and the cytoplastic Ca2+ content increased in large number.In the mature phloem ganglion,not only were there a few Ca2+ localized in the cytoplast of mature cells,but also in the differentiating cells in the vacuoles.Ca2+ was distributed in the tonoplast and vacuole contents;initiating cells almost had no Ca2+.In general,Ca2+ concentration in mature phloem ganglion cells was at a low level.The results indicated that the changes in Ca2+ distribution evoked the phloem ganglion generation,and Ca2+ regulated the physiological function of the phloem ganglion.     

短日照对休眠诱导期油桃花芽两条电子传递途径的调控

以油桃品种“曙光”为试材,采用呼吸抑制剂法研究了短日照处理下花芽在休眠诱导期两条电子传递途径的发生和运行情况.结果表明:花芽总呼吸速率(Vt)和细胞色素电子传递途径呼吸速率(ρ'Vcyt)均呈双峰曲线变化,短日照可同步诱导两者的一次峰前移、二次峰后延,抑制ρ’Vcyt,但对Vt无显著影响.交替途径容量(Valt)和实际运行活性(ρValt)亦呈双峰曲线,两者基本同步变化,短日照可以显著诱导Valt和ρValt的前期高峰期提前,提高Valt和ρValt,对后期高峰期无明显作用.细胞色素电子传递途径呼吸速率下降和交替途径呼吸速率上升是油桃花芽休眠诱导期的重要特点.从两条电子传递途径的呼吸速率对总呼吸速率的贡献率来看,细胞色素电子传递途径仍是主要电子传递途径,交替途径起辅助与分流作用.     

Cloning, tissue distribution, subcellular localization and overexpression of murine histidine-rich Ca2+ binding protein.

The histidine-rich Ca2+ binding protein (HRC) resides in the sarcoplasmic reticulum of muscle and binds Ca2+. Since Ca2+ concentrations can regulate gene expression via calcineurin, the mouse homologue of HRC (mHRC) was isolated and characterized. mHRC was detected in muscle progenitor cells, in primary clonal thymic tumors and a tumor cell line, suggesting a broader role for mHRC than in Ca2+ storage during muscle contraction. mHRC was present in the perinuclear region of myoblasts. To examine if it can regulate gene expression, mHRC was overexpressed in cells differentiating into cardiac and skeletal muscle. mHRC had no effect on cardiogenesis or myogenesis. Therefore, if mHRC plays a role in the regulation of gene expression during cellular differentiation, it does not appear to be either rate-limiting or inhibitory.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

In cardiac mitochondria, matrix free Ca²⁺ ([Ca²⁺]_m) is primarily regulated by Ca²⁺ uptake and release via the Ca²⁺ uniporter (CU) and Na⁺/Ca²⁺ exchanger (NCE) as well as by Ca²⁺ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca²⁺ buffering affects these dynamics under various Ca²⁺ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca²⁺ buffering on the uptake and release of Ca²⁺, we monitored Ca²⁺ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca²⁺] ([Ca²⁺]_e) and [Ca²⁺]_m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl₂] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca²⁺]_e and [Ca²⁺]_m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca²⁺-induced changes in mitochondrial bioenergetics. Our [Ca²⁺]_e and [Ca²⁺]_m measurements demonstrate that Ca²⁺ uptake and release do not show reciprocal Ca²⁺ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca²⁺ buffering system in the matrix compartment. The Na⁺- induced Ca²⁺ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca²⁺ uptake in the presence of large amounts of CaCl₂, but not by Na⁺- induced Ca²⁺ release.