Exogenous Ca2+ Regulation of Pollen Tube Growth and Division of Generative Nucleus in Nicotiana tabacum

Cytological and statistical studies on the effects of exogenous Ca2 + on in vitro pollen tube growth and generative nucleus (GN) division of tobacco (Nicotiana tabacum L. ) were conducted in an artificial experimental system. Under normal cultured conditions, the rate of GN division increased logarithmically in general, and reaches the climax at about 10 - 18 h. Among the treatments with various Ca2 + concentrations, 10- 3 mol/L was the optimal concentration for pollen tube growth, whereas other Ca2+ concentrations showed increasing inhibitory effect with the time of culture. Generally, Ca2 + concentrations at 10-2 and 10-3 mol/L favored GN division more than the others. Compared with 10-3 mol/L Ca2 + concentration at 10-2 mol/L benefitiated GN division at earlier stage of the treatment, but afterwards showed inhibitory effect gradually. Besides, the authors designed another series of experiments, in which 10-2, 10-1 mol/L Ca2+ (final concentrations) or 2,10 mmol/L EG-TA was respectively added to the medium containing 10-3 mol/L Ca2+ at 10 h of culture. Pollen tube growth was inhibited by the high Ca2+ treatments, especially being severely effected by 10-l mol/L Ca2 + from which wall, thickening of the tube tip, amitotic division of GN leading to micronucleus formation occurred. 10-2 mol/L Ca2 + treatment, however, promoted GN division at the earlier stage of treatment ( 10 - 12 h). EGTA treatments inhibited both pollen tube growth and GN division.     

ANTIPYRETIC ACTION OF DEXAMETHASONE ON EGTAZIC ACIDINDUCED FEVER IN RABBITS

本文用脑室灌注和Ｆｕｒａ２测定细胞内游离钙技术观察了地塞米松（ｄｅｘａｍｅｔｈａｓｏｎｅ,ＤＥＸ）对家兔乙二醇双（２氨基乙醚）四乙酸性发热效应和下丘脑细胞内游离钙浓度（［Ｃａ２＋］ｉ）的影响,借此深入探讨地塞米松解热作用的中枢机制。结果发现：脑室灌注乙二醇双（２氨基乙醚）四乙酸（０６ｎｍｏｌ）引起家兔结肠温度明显升高,静脉注射地塞米松（５ｍｇ／ｋｇ）显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,地塞米松（６０～１２０μｍｏｌ／Ｌ）并不影响下丘脑细胞内［Ｃａ２＋］ｉ,而事先脑室灌注抑制基因转录的放线菌素Ｄ（３ｎｍｏｌ）则完全取消了地塞米松对乙二醇双（２氨基乙醚）四乙酸性发热的解热作用。这些结果提示：地塞米松显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,其机制与地塞米松激活脑内某些基因的表达有关,而与下丘脑神经细胞跨膜钙离子流无关。     

Regulation of M1 Muscarinic Receptor-Mediated Signaling in Intact Cells by Exogenous,but Not Endogenously Produced,Nitric Oxide

Regulation of nitric oxide (NO) formation is critical to ensure maintenance of appropriate cellular concentrations of this labile, signaling molecule. This study investigated the role exogenous and endogenously produced NO have in feeding back to regulate NO synthesis in intact cells. Two NO donors inhibited activation of neuronal NO synthase (nNOS) in response to the muscarinic receptor agonist carbachol in Chinese hamster ovary (CHO) cells stably transfected with the M₁ muscarinic receptor and nNOS. The presence of the NO scavenger [2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide · potassium salt] (C-PTIO) potentiated carbachol-induced activation of nNOS in transfected CHO cells. C-PTIO also potentiated nNOS activity in response to the Ca²⁺ ionophore ionomycin. In contrast, the NO scavenger oxyhemoglobin depressed carbachol- and ionomycin-induced NO formation. These discrepant results suggest that it is unlikely that endogenously produced NO induces feed back inhibition at the level of nNOS activation itself. Exogenous sources of NO inhibited carbachol-induced inositol phosphates formation. However, endogenously produced NO did not appear to feed back to regulate phosphoinositide hydrolysis as there was no difference in [³H]inositol phosphates formation between cells that do or do not express nNOS. There was also no change in carbachol-induced [³H]inositol phosphates formation in the presence or absence of a NOS inhibitor or the NO scavenger C-PTIO. A decrease in the carbachol-mediated transient Ca²⁺ peak was observed in cells that express nNOS as compared to cells lacking the enzyme, suggesting that endogenous NO might inhibit receptor mediated Ca²⁺ signaling. This conclusion, however, was not supported by the lack of ability of a NOS inhibitor to modulate carbachol-induced Ca²⁺ elevations. Taken together, these results highlight differences in the regulation of the nNOS activation cascade by endogenous vs. exogenous sources of NO.     

白细胞介素-1β对骨髓基质细胞膜电位及细胞内Ca2+和pH的影响

采用荧光染料 Ｄｉ Ｂ Ａ Ｃ４（３）, Ｆｌｕｏ３／ Ａ Ｍ 和 Ｓ Ｎ Ａ Ｌ Ｆ Ｃａｌｃｅｉｎ／ Ａ Ｍ 分别标记小鼠骨髓基质细胞（ Ｂ Ｍ Ｓ Ｃ）,在激光扫描共聚焦显微镜下直接监测重组人白细胞介素１β（ Ｉ Ｌ１β）刺激后细胞膜电位,细胞内游离 Ｃａ２＋ 浓度和胞浆 ｐ Ｈ 的实时动态变化． 结果发现： Ｉ Ｌ１β加入测定体系后浓度依赖性地引起 Ｂ Ｍ Ｓ Ｃ 膜电位的迅速改变． 低浓度时发生去极化反应,高浓度时发生超极化反应． 非受体方式作用的 Ｉ Ｌ１β肽段 １６３１７１ 对膜电位无影响． Ｉ Ｌ１β不影响细胞内 Ｃａ２＋ 的浓度和胞浆ｐ Ｈ． 研究表明膜电位的变化为 Ｉ Ｌ１ 受体后早期事件,它与细胞内 Ｃａ２＋的浓度和胞浆 ｐ Ｈ 的调节无关．     

稀土离子与Ca~(2 )在生物体内的相互作用机制及应用

稀土元素在生物领域内的研究,可以分为两个方面：稀土的生物效应和稀土在其它研究中的应用．稀土元素的生物效应,主要集中在动物和医学领域．由于稀土元素的离子半径及化学性质和钙离子很相似,因此在研究稀土的生物效应时,对稀土离子与钙离子的关系颇为重视．稀土作为一种特殊的研究工具,在许多生物学研究领域中,克服了一些因其它方法的局限而不能得到的结果或简化了其中的过程．     

Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.     

Involvement of Inositol 1,4,5-Trisphosphate-Regulated Stores of Intracellular Calcium in Calcium Dysregulation and Neuron Cell Death Caused by HIV-1 Protein Tat

HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity.     

Role of calcium permeation in dihydropyridine receptor function. Insights into channel gating and excitation-contraction coupling.

The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca2+ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:2809-2816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca2+ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca2+ currents at a few potentials (from -20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca2+ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca2+ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: tau(act) = 30.7 +/- 1.9 ms, n = 11; CEIIIK: tau(act) = 2.9 +/- 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca(2+) transients with parameters of voltage dependence (V(0.5) = 6.5 +/- 3.2 mV and k = 9.3 +/- 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125-147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca2+ transients at any potential. Thus, high Ca2+ permeation is required for cardiac-type excitation-contraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to -50 mV (Qoff) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Qoff was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 +/- 2.0%, n = 6). The increase in Qoff for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Q(off) may reflect a voltage sensor movement required for activation of L-type Ca2+ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000