Cation channels in human embryonic kidney cells mediating calcium entry in response to extracellular low glucose

Glucose sensing mechanism has been intensively studied in pancreatic cells and neurons. Depolarization of membrane potential by closure of K_ATP , Kv and TASK channel, and subsequently Ca²⁺ entry via L-type voltage gated Ca²⁺ channel (VGCC) are implicated to mediate the signal transduction in these cells. However, the mechanism of non-excitable cells, which are lacking VGCC, for sensing glucose remains unclear. In this study, we utilized the calcium ratio measurement and patch clamping technique to study the effects of low glucose on [Ca²⁺]_i and currents in the human embryonic kidney epithelial cells (HEK 293). We found low glucose evoked a significant reversible [Ca²⁺]_i elevation in HEK 293 independent of the closure of Kv channels. This increase of [Ca²⁺]_i was mediated by Ca²⁺ entry across plasma membrane and exhibited a dosage dependent behaviour to external glucose concentration. The low glucose-induced entry of Ca²⁺ was characterized as a voltage independent behaviour and had cation permeability to Na⁺ and Ca²⁺. The modulation of PLC, AMPK, tyrosine kinase and cADPribose failed to regulate this glucose-sensitive Ca²⁺ entry. In addition, the entry of Ca²⁺ was insensitive to nifedipine, 2APB, SKF, La³⁺, Gd³⁺, and KBR9743, suggesting a novel signal pathway in mediating glucose sensing.     

Imaging Mitochondrial Flux in Single Cells with a FRET Sensor for Pyruvate

Mitochondrial flux is currently accessible at low resolution. Here we introduce a genetically-encoded FRET sensor for pyruvate, and methods for quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution. In HEK293 cells, neurons and astrocytes, mitochondrial pyruvate uptake was saturated at physiological levels, showing that the metabolic rate is determined by intrinsic properties of the organelle and not by substrate availability. The potential of the sensor was further demonstrated in neurons, where mitochondrial flux was found to rise by 300% within seconds of a calcium transient triggered by a short theta burst, while glucose levels remained unaltered. In contrast, astrocytic mitochondria were insensitive to a similar calcium transient elicited by extracellular ATP. We expect the improved resolution provided by the pyruvate sensor will be of practical interest for basic and applied researchers interested in mitochondrial function.     

The role of STIM1 in the receptor- and store-operated calcium influx in HEK293 cells

The possible role of STIM1 protein in the regulation of activity of receptor- and store-operated Ca²⁺ channels in non-excitable cells has been studied. Receptor- and store-operated Ca²⁺ influxes have been measured using the fluorescent method of detection of cytosolic Ca²⁺ concentration and the electrophysiological methods of whole-cell and single-channel current recordings in the control HEK293 cells and in HEK293 cells with suppressed expression of STIM1. The experiments have shown that STIM1 suppression results in a reduction of the amplitudes of both receptor- and store-operated inward calcium currents. The decrease of total Ca²⁺ influx of in response to an agonist or to passive depletion of calcium stores upon STIM1 suppression was due to the decrease or total absence of the activity of high-conductance channels I_max and non-selective channels I_ns in HEK293 cells. A decrease in the STIM1 amount also altered the activity regulation of low-conductance I_min channels that changed from exclusively agonist-operated into store-dependent channels in HEK293 cells.     

Regulation of high glucose-induced apoptosis by mitochondrial NADP+-dependent isocitrate dehydrogenase

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic kidney complications, and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidant stress. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPm activity in HEK293 cells, an embryonic kidney cell line, regulates high glucose-induced apoptosis. When we examined the protective role of IDPm against high glucose-induced apoptosis with HEK293 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPm plays an important protective role in apoptosis of HEK293 cells induced by a high concentration of glucose and may contribute to various pathologies associated with the long-term complications of diabetes.     

High glucose-induced inhibition of alpha-methyl-D-glucopyranoside uptake is mediated by protein kinase C-dependent activation of arachidonic acid release in primary cultured rabbit renal proximal tubule cells

Abnormal glucose handling in the proximal tubule may play an important role in the development of diabetic nephropathy. Thus, the present study was designed to examine the effect of high glucose on alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its signaling pathways in the primary cultured rabbit renal proximal tubule cells (PTCs). When PTCs were preincubated with 25 or 50 mM glucose for 4 h, 25 or 50 mM glucose significantly inhibited alpha-MG uptake, while 25 or 50 mM mannitol and L-glucose did not affect. Actinomycin D and cycloheximide did not block the effect of high glucose on alpha-MG uptake. Twenty-five millimoles glucose-induced inhibition of alpha-MG uptake was blocked by mepacrine and AACOCF(3), phospholipase A(2) (PLA(2)) inhibitors. Twenty-five millimoles of glucose, not mannitol or L-glucose, significantly increased the [(3)H]-arachidonic acid (AA) release compared to control. In addition, the 25 mM glucose-induced [(3)H]-AA release was completely blocked by mepacrine or AACOCF(3). Indomethacin, a cyclooxygenase inhibitor, blocked the high glucose-induced inhibition of alpha-MG uptake, although econazole, cytochrome P-450 a epoxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, did not. On the other hand, staurosporine and bisindolylmaleimide I, protein kinase C (PKC) inhibitors, blocked 25 mM glucose-induced increase of [(3)H]-AA release and inhibition of alpha-MG uptake. However, neomycin, U 73122, and phospholipase c(PLC) inhibitors did not block the effect of 25 mM glucose on [(3)H]-AA release and alpha-MG uptake. Pretreatment of methoxyverapamil, an L-type Ca(2+) channel blocker, abolished 25 mM glucose-induced increase of [(3)H]-AA release. Indeed, 25 mM glucose increased translocation of cPLA(2) from cytosolic fraction to membrane fraction. In conclusion, the present results demonstrate that high glucose inhibits alpha-MG uptake by the increase of AA release via the activation of PKC.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

Calcium inhibition and calcium potentiation of Orai1, Orai2, and Orai3 calcium release-activated calcium channels

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.     

High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 10⁴/cm² showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 10⁴/cm² largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.     

STIM1 signalling controls store-operated calcium entry required for development and contractile function in skeletal muscle

It is now well established that stromal interaction molecule 1 (STIM1) is the calcium sensor of endoplasmic reticulum stores required to activate store-operated calcium entry (SOC) channels at the surface of non-excitable cells. However, little is known about STIM1 in excitable cells, such as striated muscle, where the complement of calcium regulatory molecules is rather disparate from that of non-excitable cells. Here, we show that STIM1 is expressed in both myotubes and adult skeletal muscle. Myotubes lacking functional STIM1 fail to show SOC and fatigue rapidly. Moreover, mice lacking functional STIM1 die perinatally from a skeletal myopathy. In addition, STIM1 haploinsufficiency confers a contractile defect only under conditions where rapid refilling of stores would be needed. These findings provide insight into the role of STIM1 in skeletal muscle and suggest that STIM1 has a universal role as an ER/SR calcium sensor in both excitable and non-excitable cells.     

Role of high-voltage-activated calcium channels in glucose-regulated beta-cell calcium homeostasis and insulin release

High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, L(C)-type, and L(D)-type) in rat pancreatic beta-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.     

Lipid associated calcium ionophores in islet cell plasma membrane following glucose stimulation

The effect of glucose exposure on lipid associated calcium ionophoretic activity was measured in cultured neonatal rat pancreatic islet cells using two model systems. The first measured the ability of a lipid extract of islet cells to facilitate calcium transfer from an aqueous to organic phase and thus detected lipids which transfer calcium in the manner of authentic ionophores or which chelate the ion. In this system glucose stimulation was followed by an increase in total cell ionophoretic activity and a decrease in the activity associated with the plasma membrane. The second system measured the transfer of calcium across an artificial phospholipid membrane and detected authentic ionophoretic activity. In this model an increase in total ionophoretic activity was again seen following glucose but there was no change in the ionophoretic activity of a plasma membrane extract. The results indicate that the lipid modifications which accompany glucose-induced insulin release may alter cellular calcium stores by decreasing lipid bound calcium at the plasma membrane and increasing the capacity for calcium ionophoresis at intracellular sites.     

Rapid measurements of intracellular calcium using a fluorescence plate reader

Intracellular calcium is a universal second messenger that can serve as a broad-based measure of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-calcium levels and reduce reliance on slower, more cumbersome or expensive data collection methods. In this report, we describe a rapid and sensitive method to assay intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from multi-well plates using a fluorometer equipped with on-line injectors. We examine the compatibility of visible-light excitable dyes Calcium Green-1 and Oregon Green 488 BAPTA-1. Using this assay, we were able to detect and quantify activity from muscarinic and beta-adrenergic receptors endogenous to HEK293 cells and detect calcium signals generated by activation of Gi-coupled recombinant mu-opioid and dopamine D2L receptors, and the Gs-coupled melanocortin subtype 4 (MC4) receptor. Fluorescence signals, stable in HEK293 cells, required the use of Oregon Green 488 BAPTA-1 and an inhibitor of organic anion transport in CHO cells. Under appropriate conditions, both cell types can be used to collect complete concentration-response data for a variety of receptors (including a recombinant muscarinic M1 receptor expressed in CHO cells) from a single plate of dye-loaded cells.     

Glucocorticoid-induced changes in insulin secretion related to the metabolism and ultrastructure of pancreatic islets

The effect of adrenalectomy and dexamethasone-treatment on insulin secretion was studied and related to the changes observed in the glucose oxidation, calcium uptake, cAMP and insulin content, as well as the ultrastructure of pancreatic rat islets. It was found that adrenalectomy was followed by a decreased glucose-induced insulin secretion, glucose oxidation, calcium uptake, cAMP and insulin content without any remarkable change observed at the ultrastructural level. Conversely, adrenalectomized-rats supplemented with dexamethasone showed an increased glucose-induced insulin secretion, glucose oxidation, calcium uptake and cAMP content but a diminished islet insulin content. At the ultrastructural level, a clear picture of increased secretory activity was found, with diminished number of mature B granules and greater number of pale granules, while rough endoplasmic reticulum and Golgi complex frequently appeared hypertrophic. These changes were only observed in the B cells. On account of our results, we might suggest that insulin secretion is partially controlled by glucocorticoid circulating levels throughout their effect on pancreatic islet metabolism.     

The involvement of calcium carriers and of the vacuole in the glucose-induced calcium signaling and activation of the plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells

Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H(+)-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H(+)-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca(2+)-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H(+)-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H(+)-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP(3). Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H(+)-ATPase activation.     

12(S)-Hydroxyeicosatetraenoic acid induces cAMP production via increasing intracellular calcium concentration

We have found that a 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE), induces cAMP production in human normal fibroblast TIG-1 cells. This phenomenon was not observed in other cells tested including human embryonic kidney HEK293 cells. We have speculated that this specific response might be influenced by the kinds of isoform of adenylyl cyclase (AC) present in cells. We found that TIG-1 cells specifically expressed type VIII AC. As type VIII AC is known to be activated by an increase of calcium concentration, we determined the change of intracellular Ca2+ concentration after the addition of 12-HETE. It was elevated not only in TIG-1 cells, but also HEK293 cells, which did not respond to 12-HETE to produce cAMP. The addition of a calcium ionophore elevated the concentration of intracellular cAMP in TIG-1 cells, but it was without effect in HEK293 cells. To show that the expression of this particular isoform of AC is responsible for the positive response to 12-HETE, we transfected this AC isoform into HEK293 cells. The type VIII AC-transfected cells, in contrast to the mock-transfected ones, became very responsive to 12-HETE to produce cAMP. Taken all together the data would strongly suggest that 12-HETE specifically activates type VIII AC via increasing intracellular Ca2+ concentration.     

二氢生物喋呤还原酶参与调控HEK293T细胞自噬作用的初步研究

目的探讨二氢生物喋呤还原酶（dihydropteridine reductase,QDPR）对HEK293T细胞自噬作用的影响。方法构建野生型QDPR和突变型QDPR重组质粒分别转染HEK293T细胞,并设空载体对照组。采用RT-PCR及Western blot方法检测空载体组,野生型QDPR组和突变型QDPR组自噬相关蛋白LC3和Beclin 1的表达量变化。结果 1）测序结果证实PCR扩增得到编码正常QDPR的cDNA序列正确以及突变的cDNA也在正确的位置突变;2）磷酸钙共沉淀法转染HEK293T细胞后,野生型QDPR和突变型QDPR融合蛋白成功表达;3）RT-PCR结果显示,与对照组相比,野生型QDPR组LC3基因水平明显上调（P〈0.05）,突变型QDPR组LC3基因水平与对照组相比无统计学差异;与对照组相比,野生型和突变型组Beclin1基因水平无统计学差异;4）Western blot结果显示,与对照组相比,野生型QDPR组LC3-II和Beclin1的蛋白表达量明显上调（P〈0.05）,但LC3-I的蛋白表达量无统计学差异,突变型QDPR组与对照组相比LC3-I,II和Beclin1的蛋白表达量均没有统计学差异（P〉0.05）。结论二氢生物喋呤还原酶能增强HEK293T细胞自噬相关基因LC3和Beclin 1的表达,提示其可能具有激活自噬作用的功能;二氢生物喋呤还原酶93位氨基酸的突变影响了其对细胞自噬作用的调控,降低了自噬标志分子LC3-I和Beclin1的基因表达。     

Cytokine Stimulation Promotes Increased Glucose Uptake Via Translocation at the Plasma Membrane of GLUT1 in HEK293 Cells

Interleukin‐3 (IL‐3) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) are two of the best‐characterized cell survival factors in hematopoietic cells; these factors induce an increase in Akt activity in multiple cell lines, a process thought to be involved in cellular survival. It is known that growth factors require sustained glucose metabolism to promote cell survival. It has been determined that IL‐3 and GM‐CSF signal for increased glucose uptake in hematopoietic cells. Interestingly, receptors for IL‐3 and GM‐CSF are present in several non‐hematopoietic cell types but their roles in these cells have been poorly described. In this study, we demonstrated the expression of IL‐3 and GM‐CSF receptors in HEK293 cells and analyzed their effect on glucose uptake. In these cells, both IL‐3 and GM‐CSF, increased glucose uptake. The results indicated that this increase involves the subcellular redistribution of GLUT1, affecting glucose transporter levels at the cell surface in HEK293 cells. Also the data directly demonstrates that the PI 3‐kinase/Akt pathway is an important mediator of this process. Altogether these results show a role for non‐insulin growth factors in the regulation of GLUT1 trafficking that has not yet been directly determined in non‐hematopoietic cells. J. Cell. Biochem. 110: 1471–1480, 2010. © 2010 Wiley‐Liss, Inc.