Elevation Of Cytosolic Free Ca2 Research Articles

Difference in Contractile Mechanisms between the Early and Sustained Components of Ionomycin-Induced Contraction in Rat Caudal Arterial Smooth Muscle.

We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K+ concentration in rat caudal arterial smooth muscle involves a Ca2+-induced Ca2+ sensitization mechanism whereby Ca2+ entry through voltage-gated Ca2+ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca2+ concentration ([Ca2+]i) induced by a Ca2+ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca2+. Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC20) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca2+]i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca2+]i elevation.

Annexin 1 Is a Component of eATP-Induced Cytosolic Calcium Elevation in Arabidopsis thaliana Roots.

Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca2+ ([Ca2+]cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca2+ influx channels involved in eATP-induced [Ca2+]cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca2+ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca2+]cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes.

Single cell measurement of calpain activity in neutrophils reveals link to cytosolic Ca2+ elevation and individual phagocytotic events

It has been proposed that Ca2+ activation of calpain-1 is important for the rapid cell shape changes which accompany phagocytosis. In this paper, we use a fluorogenic calpain substrate, (CBZ-Ala Ala)2 R110, and find that there was a low calpain activity measureable in resting (ie without intentional activation) neutrophils, but that it was accelerated by an elevation of cytosolic free Ca2+ (ionomycin -induced) and inhibited by calpeptin (an established calpain-1 inhibitor). The fluorescence signal was sufficiently bright for detection in individual neutrophils that enabled the quantification of dynamic changes in calpain activity to be related to elevations in cytosolic Ca2+ within individual neutrophils. It was found that during phagocytosis of C3bi-opsonised zymosan particles, calpain activity was elevated incrementally, each step increase corresponding to the phagocytosis of an individual particle. The sub-cellular source of the fluorescent product of calpain activity was the phagocytic site itself and originated at the phagocytic cup. It was thus concluded that calpain was activated locally during the formation of the phagocytic cup. These data were consistent with central role of Ca2+ activated calpain activation in controlling phagocytosis.

C-Jun enhances intestinal epithelial restitution after wounding by increasing phospholipase C-γ1 transcription.

c-Jun is an activating protein 1 (AP-1) transcription factor and implicated in many aspects of cellular functions, but its exact role in the regulation of early intestinal epithelial restitution after injury remains largely unknown. Phospholipase C-γ1 (PLCγ1) catalyzes hydrolysis of phosphatidylinositol 4,5 biphosphate into the second messenger diacylglycerol and inositol 1,4,5 triphosphate, coordinates Ca2+ store mobilization, and regulates cell migration and proliferation in response to stress. Here we reported that c-Jun upregulates PLCγ1 expression and enhances PLCγ1-induced Ca2+ signaling, thus promoting intestinal epithelial restitution after wounding. Ectopically expressed c-Jun increased PLCγ1 expression at the transcription level, and this stimulation is mediated by directly interacting with AP-1 and CCAAT-enhancer-binding protein (C/EBP) binding sites that are located at the proximal region of the rat PLCγ1 promoter. Increased levels of PLCγ1 by c-Jun elevated cytosolic free Ca2+ concentration and stimulated intestinal epithelial cell migration over the denuded area after wounding. The c-Jun-mediated PLCγ1/Ca2+ signal also plays an important role in polyamine-induced cell migration after wounding because increased c-Jun rescued Ca2+ influx and cell migration in polyamine-deficient cells. These findings indicate that c-Jun induces PLCγ1 expression transcriptionally and enhances rapid epithelial restitution after injury by activating Ca2+ signal.

Overexpression of Selenoprotein SelK in BGC-823 Cells Inhibits Cell Adhesion and Migration.

Effects of human selenoprotein SelK on the adhesion and migration ability of human gastric cancer BGC-823 cells using Matrigel adhesion and transwell migration assays, respectively, were investigated in this study. The Matrigel adhesion ability of BGC-823 cells that overexpressed SelK declined extremely significantly (p < 0.01) compared with that of the cells not expressing the protein. The migration ability of BGC-823 cells that overexpressed SelK also declined extremely significantly (p < 0.01). On the other hand, the Matrigel adhesion ability and migration ability of the cells that overexpressed C-terminally truncated SelK did not decline significantly. The Matrigel adhesion ability and migration ability of human embryonic kidney HEK-293 cells that overexpressed SelK did not show significant change (p > 0.05) with the cells that overexpressed the C-terminally truncated protein. In addition to the effect on Matrigel adhesion and migration, the overexpression of SelK also caused a loss in cell viability (as measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) colorimetric assay) and induced apoptosis as shown by confocal microscopy and flow cytometry. The cytosolic free Ca2+ level of these cells was significantly increased as detected by flow cytometry. But the overexpression of SelK in HEK-293 cells caused neither significant loss in cell viability nor apoptosis induction. Only the elevation of cytosolic free Ca2+ level in these cells was significant. Taken together, the results suggest that the overexpression of SelK can inhibit human cancer cell Matrigel adhesion and migration and cause both the loss in cell viability and induction of apoptosis. The release of intracellular Ca2+ from the endoplasmic reticulum might be a mechanism whereby the protein exerted its impact. Furthermore, only the full-length protein, but not C-terminally truncated form, was capable of producing such impact. The embryonic cells were not influenced by the elevation of free Ca2+ level in cytosol, probably due to their much greater tolerance to the variation.

Open Stomata 1 Kinase is Essential for Yeast Elicitor-Induced Stomatal Closure in Arabidopsis.

We recently demonstrated that yeast elicitor (YEL)-induced stomatal closure requires a Ca(2+)-dependent kinase, CPK6. A Ca(2+)-independent kinase, Open Stomata 1 (OST1), is involved in stomatal closure induced by various stimuli including ABA. In the present study, we investigated the role of OST1 in YEL-induced stomatal closure in Arabidopsis using a knock-out mutant, ost1-3, and a kinase-deficient mutant, ost1-2. YEL did not induce stomatal closure or activation of guard cell S-type anion channels in the ost1 mutants unlike in wild-type plants. However, YEL did not increase OST1 kinase activity in wild-type guard cells. The YEL-induced stomatal closure and activation of S-type anion channels were also impaired in a gain-of-function mutant of a clade A type 2C protein phosphatase (ABA INSENSITIVE 1), abi1-1C. In the ost1 mutants like in the wild type, YEL induced H2O2 accumulation, activation of non-selective Ca(2+)-permeable cation (ICa) channels and transient elevations in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in guard cells. These results suggest that OST1 kinase is essential for stomatal closure and activation of S-type anion channels induced by YEL and that OST1 is not involved in H2O2 accumulation, ICa channel activation or [Ca(2+)]cyt elevations in guard cells induced by YEL.

Extracellular ATP stimulates uptake of a topoisomerase poison into cervical cancer cells

Most anticancer drugs disrupt cancer cells by interfering with vital intracellular components, such as the cell's DNA, and so must overcome the barrier posed by the plasma membrane to reach their targets. Extracellular adenosine 5'-triphosphate (ATP) can permeabilize the plasma membrane of certain cell types to relatively large ion species, through activation of P2X ion channels and through a less well-defined phospholipase C (PLC)-dependent mechanism. Using fluorescence imaging, we tested the hypothesis that extracellular ATP could stimulate uptake of fluorescent topoisomerase poisons into cervical cancer cells. Hoechst 33258 showed no evidence of uptake when applied alone, consistent with its primary use as a cell viability dye. However, subsequent co-application of ATP in the continued presence of Hoechst 33258 stimulated rapid intracellular accumulation (Fig 1A, B) via a mechanism dependent on activation of G protein-coupled P2Y receptors, PLC stimulation, and an elevation in cytosolic free Ca2+, but which was not due to a loss of immediate cell viability. Doxorubicin showed poor permeability whether applied alone or in combination with ATP. Cell viability analysis 48 hrs after a 20 min exposure to ATP and Hoechst 33258 showed that cells loaded with the toxin underwent significant cell death. ATP-evoked Hoechst 33258 uptake was observed in cultured cancer cells obtained from 5/6 sources. We did not observe ATP-evoked Hoechst 33258 uptake in healthy cervical epithelial cells obtained from the transformation zone and ectocervical tissue of 6/7 patients (Fig 1C). In summary, ATP can induce the uptake of a topoisomerase poison into cervical cancer cells without apparently causing substantial uptake into normal cervical epithelial cells. [Figure1]

Nitric oxide in guard cells as an important secondary messenger during stomatal closure.

The modulation of guard cell function is the basis of stomatal closure, essential for optimizing water use and CO2 uptake by leaves. Nitric oxide (NO) in guard cells plays a very important role as a secondary messenger during stomatal closure induced by effectors, including hormones. For example, exposure to abscisic acid (ABA) triggers a marked increase in NO of guard cells, well before stomatal closure. In guard cells of multiple species, like Arabidopsis, Vicia and pea, exposure to ABA or methyl jasmonate or even microbial elicitors (e.g., chitosan) induces production of NO as well as reactive oxygen species (ROS). The role of NO in stomatal closure has been confirmed by using NO donors (e.g., SNP) and NO scavengers (like cPTIO) and inhibitors of NOS (L-NAME) or NR (tungstate). Two enzymes: a L-NAME-sensitive, nitric oxide synthase (NOS)-like enzyme and a tungstate-sensitive nitrate reductase (NR), can mediate ABA-induced NO rise in guard cells. However, the existence of true NOS in plant tissues and its role in guard cell NO-production are still a matter of intense debate. Guard cell signal transduction leading to stomatal closure involves the participation of several components, besides NO, such as cytosolic pH, ROS, free Ca2+, and phospholipids. Use of fluorescent dyes has revealed that the rise in NO of guard cells occurs after the increase in cytoplasmic pH and ROS. The rise in NO causes an elevation in cytosolic free Ca2+ and promotes the efflux of cations as well as anions from guard cells. Stomatal guard cells have become a model system to study the signaling cascade mechanisms in plants, particularly with NO as a dominant component. The interrelationships and interactions of NO with cytosolic pH, ROS, and free Ca2+ are quite complex and need further detailed examination. While assessing critically the available literature, the present review projects possible areas of further work related to NO-action in stomatal guard cells.

Drosophila TRPML Is Required for TORC1 Activation

Loss-of-function mutations in TRPML1 (transient receptor potential mucolipin 1) cause the lysosomal storage disorder, mucolipidosis type IV (MLIV). Here, we report that flies lacking the TRPML1 homolog displayed incomplete autophagy and reduced viability during the pupal period--a phase when animals rely on autophagy for nutrients. We show that TRPML was required for fusion of amphisomes with lysosomes, and its absence led to accumulation of vesicles of significantly larger volume and higher luminal Ca(2+). We also found that trpml(1) mutant cells showed decreased TORC1 (target of rapamycin complex 1) signaling and a concomitant upregulation of autophagy induction. Both of these defects in the mutants were reversed by genetically activating TORC1 or by feeding the larvae a high-protein diet. The high-protein diet also reduced the pupal lethality and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high-protein diet caused cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between the TRPML and TORC1 pathways and raise the intriguing possibility that a high-protein diet might reduce the severity of MLIV.

Vacuolar H+-ATPase (V-ATPase) Promotes Vacuolar Membrane Permeabilization and Nonapoptotic Death in Stressed Yeast

Stress in the endoplasmic reticulum caused by tunicamycin, dithiothreitol, and azole-class antifungal drugs can induce nonapoptotic cell death in yeasts that can be blocked by the action of calcineurin (Cn), a Ca(2+)-dependent serine/threonine protein phosphatase. To identify additional factors that regulate nonapoptotic cell death in yeast, a collection of gene knock-out mutants was screened for mutants exhibiting altered survival rates. The screen revealed an endocytic protein (Ede1) that can function upstream of Ca(2+)/calmodulin-dependent protein kinase 2 (Cmk2) to suppress cell death in parallel to Cn. The screen also revealed the vacuolar H(+)-ATPase (V-ATPase), which acidifies the lysosome-like vacuole. The V-ATPase performed its death-promoting functions very soon after imposition of the stress and was not required for later stages of the cell death program. Cn did not inhibit V-ATPase activities but did block vacuole membrane permeabilization (VMP), which occurred at late stages of the cell death program. All of the other nondying mutants identified in the screens blocked steps before VMP. These findings suggest that VMP is the lethal event in dying yeast cells and that fungi may employ a mechanism of cell death similar to the necrosis-like cell death of degenerating neurons.

Systems dynamic modeling of the stomatal guard cell predicts emergent behaviors in transport, signaling, and volume control.

The dynamics of stomatal movements and their consequences for photosynthesis and transpirational water loss have long been incorporated into mathematical models, but none have been developed from the bottom up that are widely applicable in predicting stomatal behavior at a cellular level. We previously established a systems dynamic model incorporating explicitly the wealth of biophysical and kinetic knowledge available for guard cell transport, signaling, and homeostasis. Here we describe the behavior of the model in response to experimentally documented changes in primary pump activities and malate (Mal) synthesis imposed over a diurnal cycle. We show that the model successfully recapitulates the cyclic variations in H⁺, K⁺, Cl⁻, and Mal concentrations in the cytosol and vacuole known for guard cells. It also yields a number of unexpected and counterintuitive outputs. Among these, we report a diurnal elevation in cytosolic-free Ca²⁺ concentration and an exchange of vacuolar Cl⁻ with Mal, both of which find substantiation in the literature but had previously been suggested to require additional and complex levels of regulation. These findings highlight the true predictive power of the OnGuard model in providing a framework for systems analysis of stomatal guard cells, and they demonstrate the utility of the OnGuard software and HoTSig library in exploring fundamental problems in cellular physiology and homeostasis.

Plasma membrane Ca2+ transporters mediate virus‐induced acquired resistance to oxidative stress

This paper reports the phenomenon of acquired cross-tolerance to oxidative stress in plants and investigates the activity of specific Ca²+ transport systems mediating this phenomenon. Nicotiana benthamiana plants were infected with Potato virus X (PVX) and exposed to oxidative [either ultraviolet (UV-C) or H₂O₂] stress. Plant adaptive responses were assessed by the combined application of a range of electrophysiological (non-invasive microelectrode ion flux measurements), biochemical (Ca²+- and H+-ATPase activity), imaging (fluorescence lifetime imaging measurements of changes in intracellular Ca²+ concentrations), pharmacological and cytological transmission electrone microscopy techniques. Virus-infected plants had a better ability to control UV-induced elevations in cytosolic-free Ca²+ and prevent structural and functional damage of chloroplasts. Taken together, our results suggest a high degree of crosstalk between UV and pathogen-induced oxidative stresses, and highlight the crucial role of Ca²+ efflux systems in acquired resistance to oxidative stress in plants.

Protamine induces elevation of cytosolic free Ca2+ in cultured porcine aortic endothelial cells.

To test the hypothesis that protamine influences calcium movement in endothelial cells, we measured the concentration of intracellular free calcium ([Ca2+]i) in cultured porcine aortic endothelial (PAE) cells in Krebs solution (2.5mM Ca2+, pH 7.4) at 37 degrees C, by fura-2 fluorimetry. The basal [Ca2+]i of PAE cells was 113+/-18 nM (n=6). Protamine increased [Ca2+]i in a concentration-dependent manner (EC50, the concentration having 50% of the maximum effect, 1.4+/-0.3 microg mL(-1), n=6). The response of PAE cells to 100 microg mL(-1) protamine (330+/-80 nM, n=6) was blocked by a Ca2+ chelator, 5 mM glycoletherdiaminetetraacetic acid (EGTA; 131+/-16 nM, n=6), and by a non-selective Ca2+ channel blocker, 3 mM Co2+ (134+/-14 nM, n=6). These results suggest that Ca2+ influx through cell-membrane Ca2+ channels is mainly responsible for the protamine-induced Ca2+ elevation.

Carvedilol-induced elevation in cytosolic free Ca2+ level and apoptosis in SIRC corneal epithelial cells

The effect of the cardiovascular drug carvedilol on cytosolic free Ca(2+) concentrations ([Ca( 2+)](i)) and viability was examined in Statens Seruminstitut rabbit cornea (SIRC) corneal epithelial cells. [Ca(2+)](i) and cell viability were measured using the fluorescent dyes fura-2 and 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1), respectively. Carvedilol at concentrations between 1 and 30 microM increased [Ca( 2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). Carvedilol induced Mn(2+) quench of fura-2 fluorescence implicating Ca(2+) influx. The Ca(2+) influx was inhibited by suppression of protein kinase C activity. In Ca(2+)-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca( 2+) pump inhibitor), carvedilol-induced [Ca(2+)](i) rise was reduced; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca( 2+)](i) rise. Addition of the phospholipase C inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U73122; 2 microM) did not change carvedilol-induced [Ca(2+)](i) rise. At concentrations between 5 and 70 microM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 20 microM carvedilol was not reversed by prechelating cytosolic Ca(2+) with BAPTA/AM. Apoptosis was induced by 5-70 microM carvedilol. Collectively, in SIRC corneal epithelial cells, carvedilol-induced [Ca(2+)](i) rises by causing Ca(2+) release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca( 2+) influx via protein kinase C-regulated Ca(2+) channels. Carvedilol-caused cytotoxicity was mediated by Ca(2+)-independent apoptosis in a concentration-dependent manner.

Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+channels

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Elevations in cytosolic free Ca2+ are not required to trigger apoptosis in human leukaemia cells.

Previous studies have indicated that Ca2+ is a trigger for apoptosis (programmed cell death) in thymocytes and related cell lines. Recently we have shown that levels of apoptosis in leukaemic cells are diminished in Ca(2+)-deficient conditions, indicating that Ca2+ may be important in the mechanism of apoptosis in these cells. In the present study we investigated the possibility that Ca2+ serves as a trigger for apoptosis in the human leukaemic cell line, HL-60. Using fura-2 to measure cytosolic free Ca2+ concentrations, [Ca2+]i, in cell suspensions, and by using ratio imaging of fura-2 in single cells, we did not observe an early significant increase in [Ca2+]i in HL-60 cells undergoing apoptosis. The latter stages of apoptosis were, however, accompanied by increasing [Ca2+]i; these increases were apparently a result of, rather than a cause of, apoptosis. Furthermore, apoptosis could be induced in HL-60 cells under conditions of vastly reduced [Ca2+]i achieved by loading these cells with fura-2 in the presence of EGTA. These results indicate that elevation of [Ca2+]i is not a prerequisite for apoptosis in HL-60 cells and that apoptosis can occur in these cells in the presence of low [Ca2+]i.

Atrial Natriuretic Peptide Attenuates Elevations in Ca2+ and Protects Hepatocytes by Stimulating Net Plasma Membrane Ca2+ Efflux

Elevations in intracellular Ca(2+) concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca(2+) and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca(2+) rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca(2+) mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca(2+) handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca(2+). We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca(2+) into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca(2+) efflux from and inhibits ATP-stimulated Ca(2+) influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca(2+) efflux (to which both Ca(2+) efflux stimulation and Ca(2+) influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca(2+) and calpain activity. Moreover we propose that plasma membrane Ca(2+) efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca(2+)-dependent injury.

What have we learned about neuroprotection in the lab?

Abstract Purpose: Work with both in vitro and in vivo models has provided insights into how and why retinal ganglion cells (RGCs) might die in glaucoma. In vitro and in vivo models have identified mechanisms of stress or injury‐induced death of RGCs. Techniques used were cell culture, retinal whole mount and slice prepartions to more complex in vivo models. These approaches have identified activation of the alpha‐2 pathway and blockade of the NMDA receptor has important targets in neuroprotection Methods: In vitro studies used high speed confocal Ca2+ imaging at the IPL of living rat retinal slices. Changes of cytosolic free Ca2+ were monitored with the Ca2+ dye fluo‐4. Simultaneous recordings of ERG and single‐unit RGC activity were obtained from rabbit retina flat mounts. Experimental ocular hypertension was induced by laser in rats (WoldeMussie, IOVS, 2001) and primates (Hare, IOVS, 2002 Results: At the IPL, the Ca2+ signal was suppressed in a dose‐dependent manner by brimonidine and other alpha‐2 receptor agonists. The action of brimonidine was blocked by yohimbine, an alpha‐2 receptor antagonist. Application of NMDA to rabbit retinal flat mounts induced an increase in action potential spiking that was blocked by memantine. In vivo brimonidine and memantine reduced hypertensive injury to the optic nerve and retina Conclusions: These findings suggest that a function of the retinal alpha 2 system is the regulation of synaptic transmission at IPL and that brimonidine may protect RGCs by preventing abnormal elevation of cytosolic free Ca2+ either in RGCs, in their presynaptic cells, or in both. Memantine is able to block NMDA‐induced increases in in RGC spiking activity at concentrations that had little or no effect on retinal light signaling.

Protective effect of Amomi semen extract on alloxan‐induced pancreatic β‐cell damage

The protective effect of Amomi semen extract (ASE) on alloxan-induced pancreatic beta-cell damage was investigated in HIT T-15 cells, a Syrian hamster pancreatic beta-cell line. Alloxan caused pancreatic beta-cell damage through the generation of reactive oxygen species (ROS), the elevation of cytosolic free Ca2+, DNA fragmentation and the decrease of cellular NAD+ and ATP levels. All these effects of alloxan were significantly prevented by pretreatment with a water-soluble extract of Amomi semen. Pretreatment with ASE in pancreatic islets isolated from mice, also significantly abolished the inhibition of glucose-stimulated insulin secretion by alloxan. The results of this study provide evidence that ASE may have a protective activity on alloxan-induced beta-cell damage, and that the protective effect is primarily due to the inhibition of ROS generation by alloxan.

α2 Adrenergic Receptor–Mediated Modulation of Cytosolic Ca++Signals at the Inner Plexiform Layer of the Rat Retina

Compelling evidence suggests that alpha2 agonists, such as brimonidine, protect retinal ganglion cells (RGCs) from injury in a wide range of animal models. However, the mechanism of action for this protection and the physiological role of the alpha2 adrenergic system in the retina is not well understood. A major goal of this work was to explore the role of the alpha2 adrenergic system in the modulation of cytosolic Ca(2+) signaling at retinal synaptic layers, particularly the inner plexiform layer (IPL), where communication between RGCs and their presynaptic cells takes place. Functional Ca(2+) imaging at the inner plexiform layer (IPL) and outer plexiform layer (OPL) of living rat retinal slices was conducted with a high-speed confocal system. The relative changes of cytosolic free Ca(2+) were monitored with the fluorescent Ca(2+) dye fluo-4. The Ca(2+) signal was elicited by membrane depolarization produced by a high K(+) (40 mM) Ringer solution that was delivered rapidly and briefly to the test regions of the retinal slice by a custom-made multichannel local perfusion system. A brief application (8 seconds) of high K(+) Ringer elicited a robust cytosolic Ca(2+) increase at the IPL and OPL. In both cases, this Ca(2+) signal was eliminated by nimodipine, a selective L-type voltage-gated Ca(2+)-channel blocker, or when the extracellular Ca(2+) in the Ringer was replaced with equal molar EGTA. At IPL, the Ca(2+) signal was also suppressed in a dose-dependent manner by brimonidine and other alpha2 receptor agonists, such as medetomidine. The suppressive action of brimonidine and medetomidine was completely blocked by classic alpha2 receptor antagonists, such as yohimbine, rauwolscine, and atipamezole. Interestingly, the alpha2 receptor agonists had no effect on the high K(+) Ringer-elicited cytosolic Ca(2+) signal at OPL. Blocking the N-methyl-d-aspartate (NMDA) type of ionotropic glutamate receptor with D-AP5 attenuated this high K(+)-elicited Ca(2+) signal by approximately 20% at IPL. D-AP5 had no effect on the Ca(2+) signal at OPL. These findings provide the first direct evidence of alpha2 receptor-mediated modulation of L-type Ca(2+) channel activity in the CNS (the retina is part of the CNS). This alpha2 modulation appears to occur at the IPL but not at the OPL of the retina. These findings suggest that a physiological function of the retinal alpha2 system is the regulation of synaptic transmission at IPL and that brimonidine and other alpha2 agonists may protect RGCs under disease conditions by preventing abnormal elevation of cytosolic free Ca(2+) either in RGCs, in their presynaptic cells, or in both.