Role of the store-operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor-stimulated calcium oscillations in human embryonic kidney cells

We have investigated the nature of the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in human embryonic kidney (HEK293) cells by examining the roles of recently described store-operated Ca²⁺ entry proteins, Stim1 and Orai1. Knockdown of Stim1 by RNA interference (RNAi) reduced the frequency of [Ca²⁺]_i oscillations in response to a low concentration of methacholine to the level seen in the absence of external Ca²⁺. However, knockdown of Stim1 did not block oscillations in canomical transient receptor potential 3 channel (TRPC3)-expressing cells and did not affect Ca²⁺ entry in response to arachidonic acid. The effects of knockdown of Stim1 could be reversed by inhibiting Ca²⁺ extrusion with a high concentration of Gd³⁺, or by rescuing the knockdown by overexpression of Stim1. Similarly, knockdown of Orai1 abrogated [Ca²⁺]_i oscillations, and this was reversed by use of high concentrations of Gd³⁺; however, knockdown of Orai1 did not affect arachidonic acid-activated entry. RNAi targeting 34 members of the transient receptor potential (TRP) channel superfamily did not reveal a role for any of these channel proteins in store-operated Ca²⁺ entry in HEK293 cells. These findings indicate that the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in HEK293 cells depends upon the Ca²⁺ sensor, Stim1, and calcium release-activated Ca²⁺ channel protein, Orai1, and provide further support for our conclusion that it is the store-operated mechanism that plays the major role in this pathway.     

Capacitative calcium entry: from concept to molecules

Summary:  Rapid to moderately rapid changes in intracellular Ca²⁺ concentration, or Ca²⁺ signals, control a variety of critical cellular functions in the immune system. These signals are comprised of Ca²⁺ release from intracellular stores coordinated with Ca²⁺ influx across the plasma membrane. The most common mechanisms by which these two modes of signaling occur is through inositol 1,4,5-trisphosphate (IP₃)-induced release of Ca²⁺ from the endoplasmic reticulum (ER) and store-operated Ca²⁺ entry across the plasma membrane. The latter process was postulated over 20 years ago, and in just the past few years, the key molecular players have been discovered: STIM proteins serve as sensors of Ca²⁺ within the ER which communicate with and activate plasma membrane store-operated channels composed of Orai subunits. The process of store-operated Ca²⁺ entry provides support for oscillating Ca²⁺ signals from the ER and also provides direct activator Ca²⁺ that signals to a variety of downstream effectors.     

Time-dependent regulation of capacitative Ca2+ entry by IGF-1 in human embryonic kidney cells

In a wide variety of cells, mitogenic factors release Ca(2+) from intracellular stores. The fall of the [Ca(2+)] within the lumen of the Ca(2+)-storing organelles triggers in many cells capacitative Ca(2+) entry (CCE). The present study was performed to elucidate the effect of insulin-like growth factor (IGF-1) on CCE in human embryonic kidney (HEK 293) cells. After depletion of Ca(2+) stores by thapsigargin, CCE was assessed by the increase in cytosolic free [Ca(2+)] (Fura-2 fluorescence imaging) when raising extracellular [Ca(2+)] from 0 to physiological concentrations. IGF-1 exposure (50 ng/ml) for 4 h in serum-free medium markedly enhanced CCE, while a 24-h exposure to IGF-1 depressed CCE profoundly. As some Ca(2+) channels are highly sensitive to the cell membrane potential, and as IGF-1 has been reported to enhance K(+) channel activity, the influence of K(+) channel blockers on the IGF-1-dependent stimulation of CCE was also tested. TEA, charybdotoxin and margatoxin decreased CCE. Similar to the total capacitative calcium entry, the fraction of CCE that was sensitive to K(+) channel blockers was increased after 4 h and decreased after 24 h exposure to IGF-1. Taken together, these data suggest that IGF-1 induces a transient increase followed by a decrease of CCE, and that these effects are at least partly dependent on IGF-1-induced K(+) channel activity.     

Human-serum matrix supports undifferentiated growth of human embryonic stem cells

One of the most frequently used matrices for feeder-free growth of undifferentiated human embryonic stem cells (hESCs) is Matrigel, which supports attachment and growth of undifferentiated hESCs in the presence of mouse embryonic fibroblast-conditioned medium. Unfortunately, application of Matrigel or medium conditioned by mouse embryonic feeder cells is not ideal for potential medical application of hESCs because xenogeneic pathogens can be transmitted through culture conditions. We demonstrate here that human serum as matrix and medium conditioned by differentiated hESCs reduce exposure of hESCs to animal ingredients and provide a safer direction toward completely animal-free conditions for application, handling, and understanding of hESC biology. At the same time, hESCs grown under these conditions maintain all hESC features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype.     

Capacitative calcium entry is colocalised with calcium release in Xenopus oocytes: Evidence against a highly diffusible calcium influx factor

 Depletion of intracellular calcium stores activates the plasma membrane capacitative calcium entry pathway in many cell types. The nature of the signal that couples the depletion of the intracellular calcium stores to the activation of the plasma membrane calcium influx pathway is as yet unknown. It has recently been suggested that a highly diffusible calcium influx factor is involved in the activation of capacitative calcium entry, and that its action is potentiated by the protein phosphatase inhibitor okadaic acid. Depletion of intracellular calcium stores in a localised region of a Xenopus oocyte was found to evoke capacitative calcium entry exclusively colocalised across the stimulated area of the plasma membrane, arguing against the involvement of a highly diffusible calcium influx factor. Equally, no evidence could be found for the presence of a soluble calcium influx factor in the bulk cytosol of Xenopus oocytes. The potentiation of capacitative calcium entry by okadaic acid resembled that mediated by the activation of protein kinase C, thus suggesting that okadaic acid activity may not necessarily be related to the action of a putative calcium influx factor. Received: 2 January 1996 / Received after revision and accepted: 14 February 1996     

Cell cycle regulation of hepatitis C and encephalomyocarditis virus internal ribosome entry site-mediated translation in human embryonic kidney 293 cells

We have established stably transformed human embryonic kidney cell lines (HEK293) containing bicistronic constructs to study regulation of viral internal ribosome entry site (IRES)-mediated translation in vivo. These cells produce Renilla luciferase (Rluc) in a cap-dependent manner, while Firefly luciferase (Luc) synthesis is mediated by IRES elements. Using these cell lines, we demonstrate here that IRES-mediated translation directed by both hepatitis C (HCV) and encephalomyocarditis (EMCV) virus varies with the cell cycle. Experiments involving arrest of the cell lines at different phases of the cell cycle, release of synchronized cells from cell cycle arrest, as well as direct sorting of the cells based on position in the cell cycle have shown that the activity of the HCV and EMCV IRES elements is lowest during the G2/M phase in HEK293 cells. These results suggest that cellular trans-acting factors either stimulate viral IRES-mediated translation during G1 and S phases or repress translation during the G2/M phase in HEK293 cells.     

Global, synchronous oscillations in cytosolic calcium and adherence in bradykinin-stimulated Madin-Darby canine kidney cells

AIMS AND METHODS: Intercellular Ca2+ oscillations are a universal mode of signalling in both excitable and non-excitable cells. Here, we study the relationship between Ca2+ signalling and coherent changes in adhesion properties by measuring the transepithelial impedance across bradykinin-stimulated Madin-Darby canine kidney (MDCK) cell layers grown on a microelectrode. During hormone stimulation, the impedance is found to oscillate, reflecting that the cells undergo morphological/adhesive alterations with high spatio-temporal organization. The experiments are supplemented with parallel, digital imaging fluorescence microscopy of bradykinin-induced single-cell Ca2+ oscillations. RESULTS: In agreement with previous experiments, MDCK cells are found to elicit synchronous, multicellular Ca2+ oscillations in response to hormone stimulus. The periods of the Ca2+ oscillations and the electrical fluctuations are found to coincide. Further, blocking of gap junctions by 18alpha-glycyrrhetinic acid causes a loss of synchrony in Ca2+ signals and inhibition of impedance oscillations, emphasizing the importance of gap junctions in the signal transduction process. CONCLUSION: Based on these observations it is concluded that the co-ordinated adhesive changes in MDCK cells are a direct consequence of synchronized Ca2+ oscillations. Calcium signalling represents an efficient way of organizing physiological responses in a tissue. A possible functional implication of the structural changes might be to modulate transportation of various substances across the cell sheet.     

Characterization of acetylcholine- and endothelin-induced calcium entry in cultured human ciliary muscle cells

We characterized the effects of acetylcholine and endothelin on cultured human ciliary muscle cells, using the calcium-sensitive dye fura-2 to measure intracellular calcium and intracellular microelectrodes to measure the membrane potential. Both agonists, endothelin and acetylcholine, had a typcial biphasic effect on the intracellular calcium concentration. Calcium peaked initially, because of its release from intracellular stores, and then reached a plateau, owing to entry of extracellular calcium. Endothelin-induced calcium entry was almost completely blocked by addition of extracellular La³⁺ (50 mol/l) and Ni²⁺ (1 mmol/l). Acetylcholine-induced calcium entry was likewise almost completely abolished by La³⁺ and Ni²⁺. Both endothelin and acetylcholine led to an initial transient hyperpolarization with a subsequent depolarization. The hyperpolarization of the membrane potential had a time course similar to the initial calcium peak, while the depolarization occurred parallel to the calcium plateau. The depolarization induced by both agonists was reduced in the presence of La³⁺ and Ni²⁺. Verapamil (10 mol/l) had no effect on either the calcium entry or the depolarization. Acetylcholine did not induce a [Ca²⁺]_i peak when it was applied during the endothelin-induced [Ca²⁺]_i plateau and vice versa. The [Ca²⁺]_i plateau was not higher with concomitant than with single application of acetylcholine or endothelin. Thus, calcium entry and membrane depolarization induced by acetylcholine and endothelin seem to be mediated by a common La³⁺- and Ni²⁺-sensitive but verapamil-insensitive mechanism.     

Propofol activates vanilloid receptor channels expressed in human embryonic kidney 293 cells

Propofol (2,6-diisopropylphenol) is an intravenous anesthetic agent structurally unrelated to any other intravenous anesthetics. We examined the effect of propofol on a rat vanilloid receptor that was expressed in the human embryonic kidney (HEK) 293 cells by using calcium imaging method. Propofol caused a concentration-dependent increase in [Ca(2+)](i) in the HEK293 cells with the receptor. These responses were inhibited by removing extracellular calcium ions. The propofol-evoked increase in [Ca(2+)](i) in the HEK293 cells with the receptor was partially inhibited by capsazepine, a competitive antagonist of capsaicin. We conclude that propofol acts as an agonist for the receptor.     

Free calcium transients and oscillations in nerve cells

Summary Changes in the intracellular level of free calcium induced by different influences in neurones of the snail Helix pomatia have been measured by changes in Fura-2 fluorescence. Thymol in submillimolar concentrations induced the release of stored intracellular calcium. This effect was similar to xantine-induced release. IP₃ and Gpp[NH]p injections also released intracellular calcium. The response to cAMP injections was more complicated and included, probably, both the release of stored calcium and its influx through membrane channels. Oscillations of intracellular free calcium are described. It has been suggested that oscillations can occur only in cases where the mechanism of Ca-dependent calcium release is activated.     

过表达MIPU1降低阿霉素所致人胚胎肾HEK293细胞的凋亡

目的 探讨过表达MIPU1对阿霉素(Doxorubicin,DOX)所致细胞凋亡的影响及其可能的分子机制,为预防DOX抗肿瘤治疗过程中的毒副作用提供新的思路.方法 1)MTT实验观察DOX处理下过表达MIPU1对细胞存活率的影响;2)Hoechst染色和流式细胞术分析过表达MIPU1对DOX所致细胞凋亡的影响;3)RT-PCR和Westernblot分析过表达MIPU1对DOX所致Bcl-2、P53和Bax在mRNA水平和蛋白水平上表达变化的影响.结果 过表达MIPU1后,细胞存活率明显升高(P＜0.05vs pEGFP+ DOX),凋亡率显著下降(P＜0.01 vs pEGFP+ DOX),P53和Bax的表达显著下降(P＜0.05 vs pEGFP+ DOX),而Bcl-2的表达则明显增多(P＜0.05 vs pEGFP+ DOX).结论 过表达MIPU1能降低DOX引起的HEK293细胞凋亡,可能与抑制P53和Bax的表达同时促进Bcl-2的表达有关.     

Mechanical stretch regulates TRPC expression and calcium entry in human myometrial smooth muscle cells

Stretch is known to stimulate myometrial hyperplasia and hypertrophy in early pregnancy and uterine contraction at term. We propose that transduction of the stretch signal involves alteration of intracellular calcium signalling, including changes in transient receptor potential canonical (TRPC) isoform expression. The aim of the present study was to investigate the effect of prolonged mechanical (tonic) stretch in vitro on human myometrial smooth muscle cell calcium signalling and TRPC expression. Cells were cultured from myometrial biopsies, obtained from women undergoing elective Caesarean section at term, grown on Flexiplates and subjected to 25% tonic mechanical stretch for 1, 4 and 14 h. Time-matched control cells were not stretched. Mechanical stretch (14 h) increased basal calcium entry and cyclopiazonic acid (CPA)-induced calcium/Mn(2+) entry (P < 0.05) in Fura-2 loaded cells. The calcium selectivity of CPA-thapsigarin induced inward currents, measured by patch clamp electrophysiology, was also increased in stretched cells compared with control cells (P < 0.05). Real time PCR and Western blot data demonstrated that TRPC3 and TRPC4 mRNA and TRPC3 protein expression were increased by stretch (P < 0.05), respectively. These data support the hypothesis that uterine stretch modulates uterine growth and contractility in pregnancy via alterations in calcium signalling.     

An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells

Human embryonic stem cells (hESCs) have great potential as a source of cells for therapeutic uses, but their culture requires the support of mouse or human cells, either directly as a feeder cell layer or indirectly as a source of conditioned medium in feeder-free culture systems. Unfortunately, the risks of cross-transfer of pathogens from xenogeneic or allogeneic feeders or cell by-products limit their medical applications. In addition, not all human feeders support the growth of hESCs equally well, and ethical concerns have been raised regarding the derivation of feeder cells from aborted human fetuses. We report here the culture of hESCs on a novel feeder cell system, comprising fibroblast-like cells derived from the spontaneous differentiation of hESCs. Isogenicity of the hESCs and hESC-derived fibroblasts was confirmed by micro satellite analysis. The nature of the hESC-derived fibroblasts was identified by the expression of specific markers. This feeder system permits continuous growth of undifferentiated and pluripotent hESCs, as demonstrated by the expression of specific hESC markers, by the formation of teratomas after injection of hESCs into severely combined immunodeficient mice, and by in vitro differentiation of hESCs into differentiated cells of ectodermal, endodermal, and mesodermal origin. Feeder cells derived from hESCs offers a potentially more secure autogeneic and genotypically homogenous system for the growth of undifferentiated hESCs.     

Reactive blue 2 induces calcium oscillations in HeLa cells

Reactive Blue 2 (RB), which is used as an ATP receptor antagonist, induced Ca(2+) oscillations in HeLa cells. RB-induced Ca(2+) oscillations were abolished in Ca(2+)-free solution. RB, however, did not affect Ca(2+) influx measured by Mn(2+) quenching. The PLC cascade and intracellular Ca(2+) release were involved as U73122 and thapsigargin inhibited RB-induced Ca(2+) oscillations. RB enhanced a Ca(2+) response to histamine that is linked to the PLC cascade. RB may activate the PLC cascade in an extracellular Ca(2+)-dependent manner and induce Ca(2+) oscillations.     

Thrombopoietin from human embryonic kidney cells causes increased thrombocytopoiesis and decreased erythropoiesis in mice

Recent studies have shown that large doses of erythropoietin (EPO) administered daily over a 7-day period elevate erythropoiesis and lead to marked thrombocytopenia. Conversely, anaemia was found in mice following stimulation of thrombocytopoiesis by an acute thrombocytopenic episode. Although erythropoiesis and thrombocytopoiesis have been studied in mice after treatment with either hypoxia or EPO injection, only the effects of endogenous thrombopoietin (released after an acute episode of thrombocytopenia caused by an injection of antiplatelet serum) on erythropoiesis have been investigated. Therefore, we injected mice with a potent source of thrombopoietin and evaluated both thrombocytopoiesis and erythropoiesis at 3 and 5 days after treatment. The data show that thrombopoietin elevated thrombocytopoiesis with a concomitant reduction in erythropoiesis. We found significantly elevated percentage ³⁵S incorporation into platelets, platelet sizes, and total circulating platelet masses following thrombopoietin injections at both 3 and 5 days; haematocrits, reticulocyte counts, and total circulating red blood cell masses were reduced significantly in these same mice. Compared to controls treated with human serum albumin, megakaryocyte size was increased on day 3, and megakaryocyte numbers were elevated on day 5 in mice treated with thrombopoietin. Thrombopoietin did not change the blood volume of mice, but did cause an increase in splenic weight. However, splenic sequestration of red blood cells was not the cause of anaemia in mice treated with thrombopoietin, since splenectomised mice also showed increased thrombocytopoiesis with decreased erythropoiesis. These data agree with previous studies showing an inverse relation between erythropoiesis and thrombocytopoiesis, and are consistent with the hypothesis that the erythrocytic and megakaryocytic cell lines are in competition.     

Non-specific effects of calcium entry antagonists in mast cells

Calcium entry in non-excitable cells occurs through calcium-selective currents activated secondarily to store depletion and/or through non-selective cation channels (e.g., receptor- or second-messenger-activated channels). The driving force for calcium influx can be modified by chloride or potassium channels, which set the membrane potential of cells. Together, these conductances determine the extent of calcium entry. Mast cells are an excellent model system for studying calcium influx, because calcium-release-activated calcium currents (I _CRAC), second-messenger-activated non-selective currents and chloride currents are present in these cells. Whole-cell patch-clamp recordings were used to test the Effects of the commonly used calcium entry blockers econazole and SK&F 96365, as well as the antiallergic and anti-inflammatory drugs tenidap, ketotifen and cromolyn on these channels. All tested drugs blocked the three different channel types with a similar order of magnitude (IC₅₀ values ranging from micromolar to millimolar). Hence, these drugs cannot be used to discriminate between different calcium entry mechanisms.     

Roscovitine increases intracellular calcium release and capacitative calcium entry in PC12 cells

Cyclin-dependent kinase 5 (Cdk5), which is activated by the non-cyclin regulator p35 or p39, is a proline-directed serine/threonine kinase that is implicated in many physiological and pathological processes. Here, we studied calcium signaling using the fluorescent cytosolic calcium indicator, Fura-4, in NGF-differentiated PC12 cells treated with roscovitine, a Cdk5 inhibitor. As compared to the control cells, the roscovitine-treated cells significantly potentiated intracellular calcium release by membrane depolarization (high K⁺) or through thapsigargin. In addition, roscovitine increased the magnitude of capacitative calcium entry (CCE), i.e., a calcium influx mechanism triggered by the depletion of intracellular calcium stores. Notably, roscovitine markedly slowed the rate of Ca²⁺ removal from the plasma membrane. These results suggest that Cdk5 regulates intracellular calcium homeostasis and that the dysregulation of Cdk5 may contribute to disease pathogenesis by perturbing cellular Ca²⁺ signaling.     

Stage-specific antigens in human embryonic kidney and heart tissues

Summary With the aid of anaphylaxis reaction and desensitization in guinea pigs it was shown that, apart from the species and organ antigens, kidney and heart tissues of developing human embryo (6–9, 15–19, 35–40 weeks embryo) contained stage-specific antigens; in the corresponding organs of adults the latter were absent.(Presented by Acting Member of the Academy of Medical Sciences of USSR N. N. Zhukov-Verezhnikov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 57, No. 5, pp. 83–85, May, 1964.