Changes In Intracellular Calcium Research Articles

Elevated polyamines in urothelial cells from OAB subjects mediate oxotremorine-evoked rapid intracellular calcium rise and delayed acetylcholine release

Increased polyamine signaling in bladder urothelial cells (BUC) may play a role in the pathophysiology of overactive bladder (OAB). We quantitated intracellular polyamine levels in cultured BUC from OAB and asymptomatic (NB) subjects. We assessed whether polyamines modulated rapid intracellular calcium ([Ca(2+)]i) changes and delayed acetylcholine (ACh) release evoked by oxotremorine (OXO, a muscarinic agonist). BUC were cultured from cystoscopic biopsies. High-performance liquid chromatography (HPLC) quantitated intracellular putrescine, spermidine, and spermine levels. Five-millimeter difluoromethylornithine (DFMO), and one-millimeter methylglyoxalbisguanylhydrazone (MGBG) treatments were used to deplete intracellular polyamines. Ten micrometers of OXO were used to increase [Ca(2+)]i levels (measured by fura 2 microfluorimetry) and trigger extracellular ACh release (measured by ELISA). Polyamine levels were elevated in OAB compared with NB BUC (0.5 ± 0.15 vs. 0.16 ± 0.03 nmol/mg for putrescine, 2.4 ± 0.21 vs. 1.01 ± 0.13 nmol/mg for spermidine, and 1.90 ± 0.27 vs. 0.86 ± 0.26 nmol/mg for spermine; P < 0.05 for all comparisons). OXO evoked greater [Ca(2+)]i rise in OAB (205.10 ± 18.82% increase over baseline) compared with in NB BUC (119.54 ± 13.01%; P < 0.05). After polyamine depletion, OXO evoked [Ca(2+)]i rise decreased in OAB and NB BUC to 43.40 ± 6.45 and 38.82 ± 3.5%, respectively. OXO tended to increase ACh release by OAB vs. NB BUC (9.02 ± 0.1 vs. 7.04 ± 0.09 μM, respectively; P < 0.05). Polyamine depletion reduced ACh release by both OAB and NB BUC. In conclusion, polyamine levels were elevated twofold in OAB BUC. OXO evoked greater increase in [Ca(2+)]i and ACh release in OAB BUC, although these two events may be unrelated. Depletion of polyamines caused OAB BUC to behave similarly to NB BUC.

MicroRNA-145 suppresses ROS-induced Ca2+ overload of cardiomyocytes by targeting CaMKIIδ

A change in intracellular free calcium (Ca2+) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca2+ signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca2+-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H2O2-mediated Ca2+ overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca2+ overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca2+-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca2+ overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

Calcium Dynamics in Vascular Smooth Muscle

Smooth muscle cells are ultimately responsible for determining vascular luminal diameter and blood flow. Dynamic changes in intracellular calcium are a critical mechanism regulating vascular smooth muscle contractility. Processes influencing intracellular calcium are therefore important regulators of vascular function with physiological and pathophysiological consequences. In this review we discuss the major dynamic calcium signals identified and characterized in vascular smooth muscle cells. These signals vary with respect to their mechanisms of generation, temporal properties, and spatial distributions. The calcium signals discussed include calcium waves, junctional calcium transients, calcium sparks, calcium puffs, and L-type calcium channel sparklets. For each calcium signal we address underlying mechanisms, general properties, physiological importance, and regulation.

Involvement of the Orexin System in Adrenal Sympathetic Regulation

Orexin (hypocretin) is a neuropeptide secreted from hypothalamic neurons that is known to be activated during motivated behaviors and active waking. Presently, our knowledge of orexin is mainly limited to the central nervous system, and the involvement of the orexin system in peripheral tissues has received little attention. In the present study, we analyzed the existence of the orexin system in the adrenal medulla, which is part of the sympathetic nervous system. Orexin and its receptors are expressed in the bovine adrenal medulla. Orexins stimulated intracellular calcium changes and epinephrine release from cultured bovine adrenal medullary cells. Applied orexin decreased expression of prepro-orexin, orexin receptor-1 and orexin receptor-2, suggesting negative feedback regulation in the adrenal gland. Our results indicate involvement of the orexin system in the sympathetic regulation of the adrenal medulla.

Determination of a Threshold Dose to Reduce or Eliminate CdTe-Induced Toxicity in L929 Cells by Controlling the Exposure Dose

With the widespread use of quantum dots (QDs), the likelihood of exposure to quantum dots has increased substantially. The application of quantum dots in numerous biomedical areas requires detailed studies on their toxicity. In this study, we aimed to determine the threshold dose which reduced or eliminated CdTe-induced toxicity in L929 cells by controlling the exposure dose. We established a cellular model of acute exposure to CdTe QDs. Cells were exposed to different concentrations of CdTe QDs (2.2 nm and 3.5 nm) followed by illustrative cytotoxicity analysis. The results showed that low concentrations of CdTe QDs (under 10 µg/mL) promoted cell viability, caused no obvious effect on the rate of cell apoptosis, intracellular calcium levels and changes in mitochondrial membrane potential, while high concentrations significantly inhibited cell viability. In addition, reactive oxygen species in the 10 µg/mL-treated group was significantly reduced compared with the control group. In summary, the cytotoxicity of CdTe QDs on L929 cell is dose-dependent, time-dependent and size-dependent. Low concentrations of CdTe QDs (below 10 µg/mL) may be nontoxic and safe in L929 cells, whereas high concentrations (above 10 µg/mL) may be toxic resulting in inhibition of proliferation and induction of apoptosis in L929 cells.

Thromboxane A2 mediates apoptosis in cardiomyocytes via IP3

We have shown that the inflammatory mediator thromboxane A2 (TxA2) can induce ventricular arrhythmias that were associated with increased intracellular calcium in cardiomyocytes. We were able to prevent both the arrhythmias and the changes in intracellular calcium by inhibiting inositol trisphosphate (IP3) signaling. In this investigation, we sought to determine if TxA2 receptor (TxA2R) activation would trigger apoptosis in isolated adult mouse cardiomyocytes. Our hypothesis is that TxA2R‐stimulation induces IP3 formation and subsequent IP3 receptor activation triggers apoptosis. We found that TxA2Rs were expressed in the ventricles and that there was a concentration dependent increase in cell death (Trypan blue and MTT assays) following treatment with the TxA2 mimetic U46619 (0.1–10 μM). We have confirmed that TxA2‐induced death was apoptotic via cleaved caspase‐3 and TUNEL staining. Treatment of cells with the TxA2R antagonist SQ29548 and the IP3 inhibitors Gentamycin and 2‐APB eliminated the increase in apoptosis by U46619. In conclusion, our data suggests that: 1) TxA2‐induced cell death is mediated in part by apoptosis and 2) the IP3 pathway is a novel mediator of apoptosis in cardiomyocytes. These findings may provide important therapeutic targets for inflammatory‐induced cardiac apoptosis that can lead to heart failure. (AHA 11SDG5330016 to MJW)

Contribution of Intracellular Calcium and pH in Ischemic Uncoupling of Cardiac Gap Junction Channels Formed of Connexins 43, 40, and 45: A Critical Function of C-Terminal Domain

Ischemia is known to inhibit gap junction (GJ) mediated intercellular communication. However the detail mechanisms of this inhibition are largely unknown. In the present study, we determined the vulnerability of different cardiac GJ channels formed of connexins (Cxs) 43, 40, and 45 to simulated ischemia, by creating oxygen glucose deprived (OGD) condition. 5 minutes of OGD decreased the junctional conductance (Gj) of Cx43, Cx40 and Cx45 by 53±3%, 64±1% and 85±2% respectively. Reduction of Gj was prevented completely by restricting the change of both intracellular calcium ([Ca2+]i) and pH (pHi) with potassium phosphate buffer. Clamping of either [Ca2+]i or pHi, through BAPTA (2 mM) or HEPES (80 mM) respectively, offered partial resistance to ischemic uncoupling. Anti-calmodulin antibody attenuated the uncoupling of Cx43 and Cx45 significantly but not of Cx40. Furthermore, OGD could reduce only 26±2% of Gj in C-terminus (CT) truncated Cx43 (Cx43-Δ257). Tethering CT of Cx43 to the CT-truncated Cx40 (Cx40-Δ249), and Cx45 (Cx45-Δ272) helped to resist OGD mediated uncoupling. Moreover, CT domain played a significant role in determining the junction current density and plaque diameter. Our results suggest; OGD mediated uncoupling of GJ channels is primarily due to elevated [Ca2+]i and acidic pHi, though the latter contributes more. Among Cx43, Cx40 and Cx45, Cx43 is the most resistant to OGD while Cx45 is the most sensitive one. CT of Cx43 has major necessary elements for OGD induced uncoupling and it can complement CT of Cx40 and Cx45.

Butyryl- and acetylcholine-esterases in human IVF-derived follicular fluid and luteinizing granulosa cells

Background/objectives: Recent proteomic studies of human follicular fluid (FF) revealed the presence of the acetylcholine (ACh) degrading enzyme butyrylcholine-esterase (BChE). The presence of such an enzyme may be related to ACh, which is formed by human granulosa cells (GCs), and could restrict ACh-actions. ACh-actions include activation of muscarinic receptors of GCs, entailing changes in intracellular calcium, activation of transcription factors and ion channels, phosphorylation of the gap junction molecule connexin 43 and disruption of intercellular communication between GCs and proliferation. The cellular source of BChE in FF is not known. It is also unknown, if ACh-esterase (AChE), which has a variety of splice variants, is expressed in the ovary.

Functions and Imaging of Mast Cell and Neural Axis of the Gut

Close association between nerves and mast cells in the gut wall provides the microanatomic basis for functional interactions between these elements, supporting the hypothesis that a mast cell-nerve axis influences gut functions in health and disease. Advanced morphology and imaging techniques are now available to assess structural and functional relationships of the mast cell-nerve axis in human gut tissues. Morphologic techniques including co-labeling of mast cells and nerves serve to evaluate changes in their densities and anatomic proximity. Calcium (Ca(++)) and potentiometric dye imaging provide novel insights into functions such as mast cell-nerve signaling in the human gut tissues. Such imaging promises to reveal new ionic or molecular targets to normalize nerve sensitization induced by mast cell hyperactivity or mast cell sensitization by neurogenic inflammatory pathways. These targets include proteinase-activated receptor (PAR) 1 or histamine receptors. In patients, optical imaging in the gut in vivo has the potential to identify neural structures and inflammation in vivo. The latter has some risks and potential of sampling error with a single biopsy. Techniques that image nerve fibers in the retina without the need for contrast agents (optical coherence tomography and full-field optical coherence microscopy) may be applied to study submucous neural plexus. Moreover, the combination of submucosal dissection, use of a fluorescent marker, and endoscopic confocal microscopy provides detailed imaging of myenteric neurons and smooth muscle cells in the muscularis propria. Studies of motility and functional gastrointestinal disorders would be feasible without the need for full-thickness biopsy.

Investigation of Free Fatty Acid Associated Recombinant Membrane Receptor Protein Expression in HEK293 Cells Using Raman Spectroscopy, Calcium Imaging, and Atomic Force Microscopy

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

The atrazine metabolite diaminochlorotriazine suppresses LH release from murine LβT2 cells by suppressing GnRH-induced intracellular calcium transients

The primary metabolite of the herbicide atrazine (ATRA), diaminochlorotriazine (DACT), has been suggested to cause disruption in the hypothalamic-pituitary-gonadal axis leading to inhibition of luteinizing hormone (LH) release. DACT is a reactive electrophile known to form covalent protein adducts both in vitro and in vivo following ATRA exposure and maybe targeting proteins involved in GnRH-induced calcium signaling and subsequent LH release. To test this hypothesis, LβT2 pituitary cells were exposed to 300 μM DACT for 24 hrs and examined by fluorescence microscopy for GnRH-induced changes in intracellular calcium and LH release. LβT2 cells exposed to DACT had markedly diminished GnRH-induced intracellular calcium transients and a significant decreased LH release in response to GnRH. DACT appeared to cause a selective decrease in caffeine-sensitive ryanodine receptor-operated calcium stores in LβT2 cells, rather than in thapsigargin-sensitive ER calcium stores. This sensitivity correlated with the formation of covalent protein adducts by DACT, as determined by mass spectrometry. ERp57 was identified by mass spectrometry as a target of DACT adduction in the ER that could potentially mediate the effects of DACT on inhibition of GnRH-induced calcium signaling and inhibition of LH release. Intracellular calcium responses to GnRH and release of LH were restored in DACT-treated cells with the addition of a calcium ionophore (A23187). These data suggest that DACT forms adducts on proteins involved in calcium handling within the ER and that dysfunction in this critical signaling system is associated with loss of normal sensitivity to GnRH and subsequent decreased release of LH.

Mechanisms regulating resistance to inhibitors of topoisomerase II

Inhibitors of topoisomerase II (topo II) are clinically effective in the management of hematological malignancies and solid tumors. The efficacy of anti-tumor drugs targeting topo II is often limited by resistance and studies with in vitro cell culture models have provided several insights on potential mechanisms. Multidrug transporters that are involved in the efflux and consequently reduced cytotoxicity of diverse anti-tumor agents suggest that they play an important role in resistance to clinically active drugs. However, in clinical trials, modulating the multidrug-resistant phenotype with agents that inhibit the efflux pump has not had an impact. Since reduced drug accumulation per se is insufficient to explain tumor cell resistance to topo II inhibitors several studies have focused on characterizing mechanisms that impact on DNA damage mediated by drugs that target the enzyme. Mammalian topo IIα and topo IIβ isozymes exhibit similar catalytic, but different biologic, activities. Whereas topo IIα is associated with cell division, topo IIβ is involved in differentiation. In addition to site specific mutations that can affect drug-induced topo II-mediated DNA damage, post-translation modification of topo II primarily by phosphorylation can potentially affect enzyme-mediated DNA damage and the downstream cytotoxic response of drugs targeting topo II. Signaling pathways that can affect phosphorylation and changes in intracellular calcium levels/calcium dependent signaling that can regulate site-specific phosphorylation of topoisomerase have an impact on downstream cytotoxic effects of topo II inhibitors. Overall, tumor cell resistance to inhibitors of topo II is a complex process that is orchestrated not only by cellular pharmacokinetics but more importantly by enzymatic alterations that govern the intrinsic drug sensitivity.

The Preparations Used to Study Calcium in Lens Epithelial Cells and Its Role in Cataract Formation

This review summarizes relevant publications and our recent work related to the structure and the function of the lens epithelium and its role in the formation of cataract as well as its correlation with changes in intracellular calcium, as studied on different types of preparations. It is emphasized that the human lens capsule preparation isolated during the cataract surgery is an adequate source for the studies of lens epithelial cells and we highlight the possibilities for studying the intracellular calcium homeostasis and the cataract.

C‐peptide‐stimulated nitric oxide production in a cultured pulmonary artery endothelium is erythrocyte mediated and requires Zn2+

C-peptide has been shown to stimulate the production of nitric oxide (NO) in aortic endothelial cells via activation of endothelial nitric oxide synthase (eNOS) through an increased calcium influx. Here, results obtained using cultured bovine pulmonary artery endothelial cells (bPAECs) suggest that C-peptide does not induce eNOS activation directly in cultured pulmonary artery endothelium. However, C-peptide has been shown to stimulate the release of ATP from erythrocytes, a well-documented stimulus of eNOS activity in the pulmonary endothelium. Therefore, studies were performed to examine if C-peptide can indirectly stimulate NO production in a cultured pulmonary endothelium that is erythrocyte mediated. NO production and free intracellular calcium changes were monitored in immobilized bPAECs using specific intracellular fluorescent probes after stimulation with adenosine triphosphate (ATP), calcium ionophore A23187, or C-peptide. A microfluidic device enabled immobilized bPAECs to interact with flowing erythrocytes in the presence and absence of C-peptide to determine the role of the erythrocyte in C-peptide-stimulated NO production in cultured bPAECs. ATP and the calcium ionophore stimulate significant increases in both intracellular NO production and influx of free calcium in cultured bPAECs. In contrast, C-peptide, ranging from physiological to above physiological concentrations, was unable to stimulate NO production or calcium influx in the bPAECs. However, when erythrocytes were pre-incubated with a mixture containing physiological concentrations of C-peptide with Zn(2+) and haemodynamically pumped beneath bPAECs cultured on a microfluidic device, an 88.6 ± 7.5% increase in endothelial NO production was observed. C-peptide does not affect NO production in bPAECs directly but can impact NO production through an erythrocyte-mediated mechanism. Furthermore, in the absence of Zn(2+), C-peptide does not stimulate this NO production directly or indirectly. These results suggest that C-peptide, in the presence of Zn(2+), may be a determinant in purinergic receptor signalling via its ability to stimulate the release of ATP from erythrocytes.

Contribution of mitochondria to pain in diabetic neuropathy

Diabetes is a metabolic disease affecting approximately 300 million people worldwide. Neuropathy is one of its frequent complications, and may affect sensory, motor, and autonomic nerves. Its pathophysiology has not fully been elucidated. Several hypotheses have been proposed, and mitochondria have been suggested to play a significant role. This article reviews the mechanisms involved in mitochondrial dysfunction and development of diabetic neuropathy, consisting mainly of oxidative and inflammatory stress, changes in intracellular calcium regulation, apoptotic processes, and changes in mitochondrial structure and function that may lead to development of diabetic neuropathy.

Potassium Depolarization and Raised Calcium Induces α-Synuclein Aggregates

α-Synuclein is the key aggregating protein in Parkinson's disease (PD), which is characterized by cytoplasmic protein inclusion bodies, termed Lewy bodies, thought to increase longevity of the host neuron by sequestering toxic soluble α-synuclein oligomers. Previous post-mortem studies have shown relative sparing of neurons in PD that are positive for the Ca(2+) buffering protein, calbindin, and recent cell culture and in vitro studies have shown that α-synuclein aggregation can be induced by Ca(2+). We hypothesized that depolarization with potassium resulting in raised Ca(2+) in a PD cell culture model will lead to the formation of α-synuclein protein aggregates and that the intracellular Ca(2+) buffer, BAPTA-AM, may suppress their formation. Live cell fluorescence microscopy was performed to monitor changes in intracellular free calcium in HEK293T, SH-SY5Y neuroblastoma or stably transfected HEK293T/α-synuclein cells. Raised intracellular free Ca(2+) was consistently observed in cells treated with KCl, but not controls. Immunohistochemistry analysis on cells 48-72h after K(+) treatment revealed two subsets of cells with either large (>2μm), perinuclear α-synuclein aggregates or multiple smaller (<2μm), cytoplasmic accumulations. Cells pre-treated with varying concentrations of trimethadione (TMO), a calcium channel blocker, showed suppression of the Ca(2+) transient following KCl treatment and no α-synuclein aggregates at TMO concentrations >5μM. Quantitative analysis revealed a significant increase in the number of cells bearing α-synuclein cytoplasmic inclusions in both HEK293T/α-synuclein and SHSY-5Y cells when transient intracellular raised Ca(2+) was induced (p=0.001). BAPTA-AM pre-loading significantly suppressed α-synuclein aggregates (p=0.001) and the intracellular free Ca(2+) transient. This study indicates that raised intracellular Ca(2+) mediated by K(+) depolarization can lead to α-synuclein aggregation.

Inhibition of pilocarpine-induced saliva secretion by adrenergic agonists in ICR mice

The aim of the present study was to clarify the effects of the adrenoceptor agonist isoproterenol (IPR) on saliva secretion stimulated by the muscarinic receptor agonist pilocarpine (PILO) in mice. Mice were injected with either 0.5mg/kg, i.p. PILO alone or simultaneously with 2mg/kg, i.p., IPR to evaluate the inhibitory effects of adrenoceptor agonists on saliva secretion. The mechanisms underlying changes in saliva flow rate were evaluated by histological examination of aquaporin 5 (AQP5) and saliva flow rate using the adenylate cyclase (AC) inhibitor SQ22536 (0.25mg per mouse, s.c.), which was administered 30min prior to PILO and/or IPR. Saliva volume decreased significantly in the mice treated simultaneously with PILO+IPR compared with that in mice treated with PILO alone. Changes in the intracellular localization of AQP5 were seen in PILO+IPR-treated mice, and those changes were reversed by SQ22536 pretreatment. In addition, the decreased salivary flow rate in the PILO+IPR-treated mice was partially restored by SQ22536 pretreatment. There were no significant changes in intracellular calcium or ATP levels among the groups. The results of the present study suggest the existence of an inhibitory effect of the sympathetic nervous system on parasympathetic-stimulated salivary secretion from the salivary gland.

Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells

Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague–Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.

Metabolomic analysis of pancreatic beta cells following exposure to high glucose

BackgroundChronic exposure to hyperglycaemic conditions has been shown to have detrimental effects on beta cell function. The resulting glucotoxicity is a contributing factor to the development of type 2 diabetes. The objective of this study was to combine a metabolomics approach with functional assays to gain insight into the mechanism by which glucotoxicity exerts its effects. MethodsThe BRIN-BD11 and INS-1E beta cell lines were cultured in 25mM glucose for 20h to mimic glucotoxic effects. PDK-2 protein expression, intracellular glutathione levels and the change in mitochondrial membrane potential and intracellular calcium following glucose stimulation were determined. Metabolomic analysis of beta cell metabolite extracts was performed using GC–MS, 1H NMR and 13C NMR. ResultsConditions to mimic glucotoxicity were established and resulted in no loss of cellular viability in either cell line while causing a decrease in insulin secretion. Metabolomic analysis of beta cells following exposure to high glucose revealed a change in amino acids, an increase in glucose and a decrease in phospho-choline, n−3 and n−6 PUFAs during glucose stimulated insulin secretion relative to cells cultured under control conditions. However, no changes in calcium handling or mitochondrial membrane potential were evident. ConclusionsResults indicate that a decrease in TCA cycle metabolism in combination with an alteration in fatty acid composition and phosphocholine levels may play a role in glucotoxicity induced impairment of glucose stimulated insulin secretion. General significanceAlterations in certain metabolic pathways play a role in glucotoxicity in the pancreatic beta cell.

FK506 Induced Apoptosis Is Mediated by Endoplasmic Reticulum Derived Calcium Dependent Caspase-12 in Jurkat Cells

Purpose: The effect of FK506 on endoplasmic reticulum(ER) derived calcium and caspase-12 mediated apoptosis in Jurkat human T-lymphocyte was investigated. Method: Cell viability was measured by flow cytometry. Intracellular calcium generation was measured. Western blotting of procaspase-12 was performed. And the catalytic activity of caspase -3, -6, -8, and -9 proteases in Jurkat cells was also measured. Results: FK506 dose-dependently decreased the viability in Jurkat cells. Increased intracellular accumulation of calcium in FK506 treated Jurkat cells from 24hours. FK506 continuously increased calcium concentration from 24hours to 72hrs. There was no evidence of calcium ionorpore (A23187) increased intracellular calcium changes with or without FK506 but calcium ATPase inhibitor (Thapsigargin) increased intracellular calcium accumulation and FK506 more and more increased calcium ATPase inhibitor(Thapsigargin) derived intracellular calcium accumulation. Procaspase-12 protease analyzed from 48hours. Treatment of cells with FK506 increased activation of caspase-12 protease. FK506 increased the catalytic activity of caspase-3 but there was no evidence of increased catalytic activation of caspase-6, -8 and -9 proteases in Jurkat cells. Conclusion: These data indicate that understanding of ER derived calcium and caspase-12 mediated apoptosis in human Jurkat cell line.