Characterization of pollen tube development in Pinus strobus (Eastern white pine) through proteomic analysis of differentially expressed proteins

The differentially expressed proteins in pollen tubes indicate their specific roles in this stage of male gametophyte development. To isolate these proteins, 2-DE was done using ungerminated pollen and 2-day-old pollen tubes of Pinus strobus. Results show that 645 and 647 protein spots were clearly resolved from pollen grains and pollen tubes, respectively. Thirty-eight protein spots were expressed only in pollen tubes, while 19 increased in intensity. MALDI-TOF MS was used to generate tryptic peptide masses that were submitted to Mascot for identification. Of the differentially expressed proteins, 12% matched with hypothetical proteins, 33% did not hit any protein, and for the 55%, a putative function was assigned based on similarity of sequences with previously characterized proteins. Therefore, pollen tube development can be characterized by the cellular activities that involve metabolism, stress/defense response, gene regulation, signal transduction, and cell wall formation. This study expands our understanding of the changes in protein expression associated with pollen tube development and provides insights into the molecular programs that separate the development of the pollen tubes from pollen grains. This is the first report that describes a global analysis of differentially expressed proteins from the pollen tube of any seed plant.     

Inhibition of RNA and protein synthesis in pollen tube development of Pinus bungeana by actinomycin D and cycloheximide

* The effects of actinomycin D and cycloheximide on RNA and protein synthesis were investigated during pollen tube development of Pinus bungeana. * RNA and protein contents, protein expression patterns, cell wall components and ultrastructural changes of pollen tubes were studied using spectrophotometry, SDS-PAGE electrophoresis, Fourier transformed infrared (FTIR) microspectroscopy and transmission electron microscopy (TEM). * Pollen grains germinated in the presence of actinomycin D, but tube elongation and RNA synthesis were inhibited. By contrast, cycloheximide inhibited pollen germination and protein synthesis, induced abnormal tube morphology, and retarded the tube growth rate. SDS-PAGE analysis showed that protein expression patterns changed distinctly, with some proteins being specific for each phase. FTIR microspectroscopy established significant changes in the chemical composition of pollen tube walls. TEM analysis revealed the inhibitors caused disintegration of organelles involved in the secretory system. * These results suggested RNA necessary for pollen germination and early tube growth were present already in the pollen grains before germination, while the initiation of germination and the maintenance of pollen tube elongation depended on continuous protein synthesis.     

2-aminoethoxydiphenyl borate reveals heterogeneity in receptor-activated Ca(2+) discharge and store-operated Ca(2+) influx.

We have investigated Ca(2+) release and receptor- and store-operated Ca(2+) influxes in Chinese hamster ovary-K1 (CHO) cells, SH-SY5Y human neuroblastoma cells and RBL-1 rat basophilic leukemia cells using Fura-2 and patch-clamp measurements. Ca(2+) release and subsequent Ni(2+)-sensitive, store-operated influx were induced by thapsigargin and stimulation of G protein-coupled receptors. The alleged noncompetitive IP3 receptor inhibitor,2-aminoethoxydiphenyl borate (2-APB) rapidly blocked a major part of the secondary influx response in CHO cells in a reversible manner. It also reduced Mn(2+) influx in response to thapsigargin. Inhibition of Ca(2+) release was also seen but this was less complete, slower in onset, less reversible, and required higher concentration of 2-APB. In RBL-1 cells, I(CRAC) activity was rapidly blocked by extracellular 2-APB whereas intracellular 2-APB was less effective. Store-operated Ca(2+) influxes were only partially blocked by 2-APB. In SH-SY5Y cells, Ca(2+) influxes were insensitive to 2-APB. Ca(2+) release in RBL-1 cells was partially sensitive but in SH-SY5Y cells the release was totally resistant to 2-APB. The results suggest, that 2-APB (1) may inhibit distinct subtypes of IP3 receptors with different sensitivity, and (2) that independently of this, it also inhibits some store-operated Ca(2+) channels via a direct, extracellular action.     

Arachidonic acid in astrocytes blocks Ca(2+) oscillations by inhibiting store-operated Ca(2+) entry,and causes delayed Ca(2+) influx

ATP-elicited oscillations of the concentration of free intracellular Ca(2+) ([Ca(2+)](i)) in rat brain astrocytes were abolished by simultaneous arachidonic acid (AA) addition, whereas the tetraenoic analogue 5,8,11,14-eicosatetraynoic acid (ETYA) was ineffective. Inhibition of oscillations is due to suppression by AA of intracellular Ca(2+) store refilling. Short-term application of AA, but not ETYA, blocked Ca(2+) influx, which was evoked by depletion of stores with cyclopiazonic acid (CPA) or thapsigargin (Tg). Addition of AA after ATP blocked ongoing [Ca(2+)](i) oscillations. Prolonged AA application without or with agonist could evoke a delayed [Ca(2+)](i) increase. This AA-induced [Ca(2+)](i) rise developed slowly, reached a plateau after 5 min, could be reversed by addition of bovine serum albumin (BSA), that scavenges AA, and was blocked by 1 microM Gd(3+), indicative for the influx of extracellular Ca(2+). Specificity for AA as active agent was demonstrated by ineffectiveness of C16:0, C18:0, C20:0, C18:2, and ETYA. Moreover, the action of AA was not affected by inhibitors of oxidative metabolism of AA (ibuprofen, MK886, SKF525A). Thus, AA exerted a dual effect on astrocytic [Ca(2+)](i), firstly, a rapid reduction of capacitative Ca(2+) entry thereby suppressing [Ca(2+)](i) oscillations, and secondly inducing a delayed activation of Ca(2+) entry, also sensitive to low Gd(3+) concentration.     

Ca(2+) influx and neurotransmitter release at ribbon synapses

Ca(2+) influx through voltage-gated Ca(2+) channels triggers the release of neurotransmitters at presynaptic terminals. Some sensory receptor cells in the peripheral auditory and visual systems have specialized synapses that express an electron-dense organelle called a synaptic ribbon. Like conventional synapses, ribbon synapses exhibit SNARE-mediated exocytosis, clathrin-mediated endocytosis, and short-term plasticity. However, unlike non-ribbon synapses, voltage-gated L-type Ca(2+) channel opening at ribbon synapses triggers a form of multiquantal release that can be highly synchronous. Furthermore, ribbon synapses appear to be specialized for fast and high throughput exocytosis controlled by graded membrane potential changes. Here we will discuss some of the basic aspects of synaptic transmission at different types of ribbon synapses, and we will emphasize recent evidence that auditory and retinal ribbon synapses have marked differences. This will lead us to suggest that ribbon synapses are specialized for particular operating ranges and frequencies of stimulation. We propose that different types of ribbon synapses transfer diverse rates of sensory information by expressing a particular repertoire of critical components, and by placing them at precise and strategic locations, so that a continuous supply of primed vesicles and Ca(2+) influx leads to fast, accurate, and ongoing exocytosis.     

Contribution of endoplasmic reticulum to Ca(2+) signals in Dictyostelium depends on extracellular Ca(2+)

We recently reported the first molecular genetic evidence that Dictyostelium Ca²⁺ responses to chemoattractants include a contribution from the endoplasmic reticulum (ER) – responses are enhanced in mutants lacking calreticulin or calnexin, two major Ca²⁺-binding proteins in the ER, even though the influx of Ca²⁺ into the mutants is reduced. Compared with wild-type cells, the ER in the mutants contributes at least 30–70 nM additional Ca²⁺ to the responses. Here we report that this additional ER contribution to the cytosolic Ca²⁺ signal depends upon extracellular Ca²⁺– it does not occur in the absence of extracellular Ca²⁺, increases to a maximum as the extracellular Ca²⁺ levels rise to 10 μM and then remains constant at extracellular Ca²⁺ concentrations up to at least 250 μM. These results suggest that Ca²⁺ influx causes the intracellular release, in the simplest scenario by a mechanism involving Ca²⁺-induced Ca²⁺ release from the ER. By way of contrast, we show that Ca²⁺ responses to mechanical stimulation are reduced, but still occur in the absence of extracellular Ca²⁺. Unlike the responses to chemoattractants, mechanoresponses thus include contributions from the ER that are independent of extracellular Ca²⁺.     

Extracellular Ca(2+) influx and NO generation are inhibited by small interference RNA targeting extracellular Ca(2+)-sensing receptor in human umbilical vein endothelial cells

To investigate the effect of Ca(2+)-sensing receptor (CaR) on Spermine-induced extracellular Ca(2+) influx and NO generation in human umbilical vein endothelial cells (HUVEC), the small interference RNA (siRNA) specifically targeting CaR gene was designed, synthesized and transfected into HUVEC according to the cDNA sequence of human CaR gene in GenBank. The transfection efficiency and the interference efficiency of CaR protein were determined by laser scanning confocal microscopy and Western blot, respectively. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by Fura-2/AM loading. The production of NO and the activity of endothelial nitric oxide synthase (eNOS) were determined by the DAF-FM diacetate (DAF-FM DA). Western blot results demonstrated that siRNA targeting the CaR specifically decreased the expression of CaR protein in CaR siRNA group 48 h after transfection (P < 0.05). At the same time, the Spermine-induced [Ca(2+)](i), eNOS activity and NO generation were also significantly reduced (P < 0.05) in CaR siRNA group compared with those in the untransfected or negative siRNA transfected group. In conclusion, the present study suggests that the CaR plays an important role in the Spermine-evoked process of extracellular Ca(2+) influx and NO generation in HUVEC.     

Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells

The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca(2+)-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca(2+) homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca(2+) leak (while leaving Ca(2+) accumulation unaffected), hence reducing the steady-state [Ca(2+)] levels. As a direct consequence, the [Ca(2+)] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca(2+) influx activated by Ca(2+) store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca(2+)-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca(2+) homeostasis may contribute to the protective role of this oncogene against programmed cell death.     

小凹蛋白-1下调人脐静脉内皮细胞外钙敏感受体介导的钙内流

为了探讨小凹蛋白-1(caveolin-1,Cav-1)在人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)细胞外钙敏感受体(extracellular Ca2+-sensing receptor,CaR)介导Ca2+内流中的作用,本实验研究了细胞膜穴样凹陷(caveolae)结构破坏剂Filipin或Cav-1基因沉默后对CaR介导Ca2+内流的影响。Fura-2/AM负载检测细胞内Ca2+浓度(intracellular Ca2+ concentration,[Ca2+]i)。结果显示,HUVECs中CaR对不同浓度细胞外Ca2+刺激无反应。无论细胞外为零钙液或含钙液时,精胺(Spermine,2mmol/L)刺激CaR时均引起[Ca2+]i升高(P<0.05),其中细胞外液为含钙液时,[Ca2+]i升高较细胞外为零钙液时更明显(P<0.05),CaR的负性变构调节剂Calhex231(1μmol/L)均可完全阻断Spermine刺激引起的[Ca2+]i升高(P<0.05);相反,Spermine升高[Ca2+]i作用可被Filipin(1.5μ...     

A Xestospongin C-sensitive Ca(2+) store is required for cAMP-induced Ca(2+) influx and cAMP oscillations in Dictyostelium

Xestospongin C (XeC) is known to bind to the inositol 1,4, 5-trisphosphate (IP(3))-sensitive store in mammalian cells and to inhibit IP(3)- and thapsigargin-induced Ca(2+) release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca(2+) uptake into purified vesicle fractions and induced Ca(2+) release. This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP(3) receptor channel as was proposed for mammalian cells (Gafni, J., Munsch, J. A. , Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723-733). In order to elucidate the function of the XeC-sensitive Ca(2+) store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca(2+)](i) and inhibited cAMP-induced Ca(2+) influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.     

Excitation-induced Ca(2+) influx in rat soleus and EDL muscle: mechanisms and effects on cellular integrity

In rat skeletal muscle, electrical stimulation increases Ca(2+) influx leading to progressive accumulation of calcium. Excitation-induced Ca(2+) influx in extensor digitorum longus (EDL; fast-twitch fibers) and soleus muscle (slow-twitch fibers) is compared. In EDL and soleus, stimulation at 40 Hz increased (45)Ca uptake 34- and 21-fold and (22)Na uptake 17- and 7-fold, respectively. These differences may be related to the measured 70% higher concentration of Na(+) channels in EDL. Repeated stimulation at 40 Hz elicited a delayed release of lactic acid dehydrogenase (LDH) from EDL (11-fold increase) and soleus (5-fold increase). Continuous stimulation at 1 Hz increased LDH release only from EDL (18-fold). This was associated with increased Ca(2+) content and was augmented at high extracellular Ca(2+) concentration ([Ca(2+)](o)) and suppressed at low [Ca(2+)](o). The data support the hypothesis that excitation-induced Ca(2+) influx is mediated in part by Na(+) channels and that the ensuing increase in intracellular Ca(2+) induces cellular damage. This is most pronounced in EDL, which may account for the repeated observation that prolonged exercise leads to preferential damage to fast-twitch fibers.     

Ca(2+) influx and opening of Ca(2+)-activated K(+) channels in muscle fibers from control and mdx mice

Using the patch-clamp technique, we demonstrate that, in depolarized cell-attached patches from mouse skeletal muscle fibers, a short hyperpolarization to resting value is followed by a transient activation of Ca(2+)-activated K(+) channels (K(Ca)) upon return to depolarized levels. These results indicate that sparse sites of passive Ca(2+) influx at resting potentials are responsible for a subsarcolemmal Ca(2+) load high enough to induce K(Ca) channel activation upon muscle activation. We then investigate this phenomenon in mdx dystrophin-deficient muscle fibers, in which an elevated Ca(2+) influx and a subsequent subsarcolemmal Ca(2+) overload are suspected. The number of Ca(2+) entry sites detected with K(Ca) was found to be greater in mdx muscle. K(Ca) activity reflecting subsarcolemmal Ca(2+) load was also found to be independent of the activity of leak channels carrying inward currents at negative potentials in mdx muscle. These results indicate that the sites of passive Ca(2+) influx newly described in this study could represent the Ca(2+) influx pathways responsible for the subsarcolemmal Ca(2+) overload in mdx muscle fibers.     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes

The effects of Mg(2+) on reactive oxygen species (ROS) and cell Ca(2+) during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2',7'-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H(2)O(2) and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O(2)(-).), respectively. Rapidly exchangeable cell Ca(2+) was determined by (45)Ca(2+) uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100-130% during reoxygenation alone and further increased 60% by increasing extracellular Mg(2+) concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16-24% during reoxygenation but were not affected by Mg(2+). Cell Ca(2+) increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg(2+), 57% by 10 microM 3,4-dichlorobenzamil (DCB) (inhibitor of Na(+)/Ca(2+) exchange), and 75% by combining Mg(2+) and DCB. H(2)O(2) (25 and 500 microM) reduced Ca(2+) accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso-N-acetyl-penicillamine (1 mM) had no effect. Mg(2+) reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg(2+) to 5 mM increased the fluorescence of the H(2)O(2)/NO-sensitive probe DCF without increasing that of the O(2)(-).-sensitive probe ETH, reduced Ca(2+) accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg(2+), may be linked to the effect of Mg(2+) on Ca(2+) accumulation and/or ROS levels.     

Molecular evolution and structural analysis of the Ca(2+) release-activated Ca(2+) channel subunit, Orai

Depletion of intracellular Ca(2+) stores evokes Ca(2+) entry across the plasma membrane by inducing Ca(2+) release-activated Ca(2+) (CRAC) currents in many cell types. Recently, Orai and STIM proteins were identified as the molecular identities of the CRAC channel subunit and the endoplasmic reticulum Ca(2+) sensor, respectively. Here, extensive database searching and phylogenetic analysis revealed several lineage-specific duplication events in the Orai protein family, which may account for the evolutionary origins of distinct functional properties among mammalian Orai proteins. Based on similarity to key structural domains and essential residues for channel functions in Orai proteins, database searching also identifies a putative primordial Orai sequence in hyperthermophilic archaeons. Furthermore, modern Orai appears to acquire new structural domains as early as Urochodata, before divergence into vertebrates. The evolutionary patterns of structural domains might be related to distinct functional properties of Drosophila and mammalian CRAC currents. Interestingly, Orai proteins display two conserved internal repeats located at transmembrane segments 1 and 3, both of which contain key amino acids essential for channel function. These findings demonstrate biochemical and physiological relevance of Orai proteins in light of different evolutionary origins and will provide novel insights into future structural and functional studies of Orai proteins.     

Ca(2+) measurements in skinned cardiac fibers: effects of Mg(2+) on Ca(2+) activation of force and fiber ATPase.

In contrast to previous studies, a new fluorescent method was used to accurately determine the Ca(2+) concentration in test solutions used to activate skinned rat cardiac cells. This method used the calcium green-2 fluorescent indicator, which is shown to change its fluorescence over the Ca(2+) range responsible for Ca(2+) activation of force and ATPase. The dissociation constant (K(d)) of calcium green-2 for Ca(2+) was determined for three different Mg(2+) concentrations in solutions similar to those used in the experiment. Increasing Mg(2+) concentration from 1.0 to 8.0 mM had no significant effect on the Ca(2+) sensitivity of either force or actomyosin ATPase activity, in contrast to previous reported studies on force. The ATPase activity was activated at lower Ca(2+) concentration than the force. The ratio (ATPase/force) is proportional to the dissociation rate of force-generating myosin cross bridges and decreased during Ca(2+) activation. These findings are consistent with the hypothesis that cardiac muscle contraction is activated by a single Ca(2+)-specific binding site on troponin C.     

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.