Glutamate-induced exocytosis of glutamate from astrocytes

Recent studies indicate that astrocytes can play a much more active role in neuronal circuits than previously believed, by releasing neurotransmitters such as glutamate and ATP. Here we report that local application of glutamate or glutamine synthetase inhibitors induces astrocytic release of glutamate, which activates a slowly decaying transient inward current (SIC) in CA1 pyramidal neurons and a transient inward current in astrocytes in hippocampal slices. The occurrence of SICs was accompanied by an appearance of large vesicles around the puffing pipette. The frequency of SICs was positively correlated with [glutamate]o. EM imaging of anti-glial fibrillary acid protein-labeled astrocytes showed glutamate-induced large astrocytic vesicles. Imaging of FM 1-43 fluorescence using two-photon laser scanning microscopy detected glutamate-induced formation and fusion of large vesicles identified as FM 1-43-negative structures. Fusion of large vesicles, monitored by collapse of vesicles with a high intensity FM 1-43 stain in the vesicular membrane, coincided with SICs. Glutamate induced two types of large vesicles with high and low intravesicular [Ca2+]. The high [Ca2+] vesicle plays a major role in astrocytic release of glutamate. Vesicular fusion was blocked by infusing the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or the SNARE blocker, tetanus toxin, suggesting Ca2+- and SNARE-dependent fusion. Infusion of the vesicular glutamate transport inhibitor, Rose Bengal, reduced astrocytic glutamate release, suggesting the involvement of vesicular glutamate transports in vesicular transport of glutamate. Our results demonstrate that local [glutamate]o increases induce formation and exocytotic fusion of glutamate-containing large astrocytic vesicles. These large vesicles could play important roles in the feedback control of neuronal circuits and epileptic seizures.     

Cyclooxygenase products stimulate carbon monoxide production by piglet cerebral microvessels

Products of arachidonic acid (AA) metabolism by cyclooxygenase (Cox) are important in regulation of neonatal cerebral circulation. The brain and cerebral microvessels also express heme oxygenase (HO) that metabolizes heme to carbon monoxide (CO), biliverdin, and iron. The purpose of this study in newborn pig cerebral microvessels was to address the hypothesis that Cox products affect HO activity and HO products affect Cox activity. AA (2.0-20 microM) increased prostaglandin E2 (PGE2) measured by radioimmunoassay (RIA) and also CO measured by gas chromatography/mass spectrometry (GC/MS). Further, 10(-4) M indomethacin, which inhibited Cox, reduced both AA and heme-induced CO production. Conversely, neither exogenous 2 x 10(-6) M heme, which markedly increased CO production, nor the inhibitor of HO, chromium mesoporphyrin, altered PGE2 synthesis. Because AA metabolism by Cox generates both prostanoids and superoxides, we determined the effects of the predominant prostanoid and superoxide on CO production. Although PGE2 caused a small increase in CO production, xanthine oxidase plus hypoxanthine, which produces superoxide, strongly stimulated the production of CO by cerebral microvessels. This increase was mildly attenuated by catalase. These data suggest that Cox-catalyzed AA metabolites, most likely superoxide and/or a subsequent reactive oxygen species, increase cerebrovascular CO production. This increase seems to be caused, at least in part, by the elevation of HO-2 catalytic activity. Conversely, Cox activity is not affected by HO-catalyzed heme metabolites. These data suggest that some cerebrovascular functions attributable to Cox activity could be mediated by CO.     

Endothelins stimulate the production of stromelysin-1 in cultured rat astrocytes

The effects of endothelins (ETs) on the production of stromelysins, a sub-family of matrix metalloproteinases, were examined in cultured astrocytes. The treatment of cultured rat astrocytes with ET-1 increased stromelysin-1 mRNA levels, while stromelysin-2 and -3 mRNAs were not affected. Immunocytochemical observations showed that cultured astrocytes produced stromelysin-1 protein. ET-1 and Ala^1,3,11,15-ET-1, an ET_B receptor selective agonist, stimulated the release of stromelysin-1 from cultured astrocytes. Accompanying the increase in protein release, the peptidase activity of stromelysin-1 in the medium was also increased by ET-1. The effects of ET-1 on astrocytic stromelysin-1 expression were inhibited by PD98059, staurosporine, and Ca²⁺ chelation, but not by SB203580 or pyrrolidine dithiocarbamate. These results show that activation of astrocytic ET receptors stimulates the production of stromelysin-1, suggesting a role for ETs in stromelysin production in brain pathologies.     

Glutamate-induced swelling of cultured astrocytes is mediated by metabotropic glutamate receptor

The effects of glutamate and its agonists and antagonists on the swelling of cultured astrocytes were studied. Swelling of astrocytes was measured by [3H]-O-methyl-D-glucose uptake. Glutamate at 0.5, 1 and 10mmol/L and irons-l-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), a metabotropic glutamate receptor (mGluR) agonist, at 1 mmol/L caused a significant increase in astrocytic volume, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) was not effective. L-2-amino-3-phosphonopropionic acid (L-AP3), an antagonist of mGluR, blocked the astrocytic swelling induced by trans-ACPD or glutamate. In Ca2+-free condition, glutamate was no longer effective. Swelling of astrocytes induced by glutamate was not blocked by CdCl2 at 20 μmol/L, but significantly reduced by CdCl2 at 300 μmol/L and dantrolene at 30 μmol/L. These findings indicate that mGluR activation results in astrocytic swelling and both extracellular calcium and internal calcium stores play important roles in the genes     

Beta-amyloid peptides stimulate endozepine biosynthesis in cultured rat astrocytes

Accumulation of beta-amyloid peptide (Abeta), which is a landmark of Alzheimer's disease, may alter astrocyte functions before any visible symptoms of the disease occur. Here, we examined the effects of Abeta on biosynthesis and release of diazepam-binding inhibitor (DBI), a polypeptide primarily expressed by astroglial cells in the CNS. Quantitative RT-PCR and specific radioimmunoassay demonstrated that aggregated Abeta(25-35), at concentrations up to 10(-4) m, induced a dose-dependent increase in DBI mRNA expression and DBI-related peptide release from cultured rat astrocytes. These effects were totally suppressed when aggregation of Abeta(25-35) was prevented by Congo red. Measurement of the number of living cells revealed that Abeta(25-35) induced a trophic rather than a toxic effect on astrocytes. Administration of cycloheximide blocked Abeta(25-35)-induced increase of DBI gene expression and endozepine accumulation in astrocytes, indicating that protein synthesis is required for DBI gene expression. Altogether, the present data suggest that Abeta-induced activation of endozepine biosynthesis and release may contribute to astrocyte proliferation associated with Alzheimer's disease.     

Glutamate-induced deregulation of calcium homeostasis and mitochondrial dysfunction in mammalian central neurones

Delayed neuronal death following prolonged (10-15 min) stimulation of Glu receptors is known to depend on sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) which may persist far beyond the termination of Glu exposure. Mitochondrial depolarization (MD) plays a central role in this Ca(2+) deregulation: it inhibits the uniporter-mediated Ca(2+) uptake and reverses ATP synthetase which enhances greatly ATP consumption during Glu exposure. MD-induced inhibition of Ca(2+) uptake in the face of continued Ca(2+) influx through Glu-activated channels leads to a secondary increase of [Ca(2+)](i) which, in its turn, enhances MD and thus [Ca(2+)](i). Antioxidants fail to suppress this pathological regenerative process which indicates that reactive oxygen species are not involved in its development. In mature nerve cells (>11 DIV), the post-glutamate [Ca(2+)](i) plateau associated with profound MD usually appears after 10-15 min Glu (100 microM) exposure. In contrast, in young cells (<9 DIV) delayed Ca(2+) deregulation (DCD) occurs only after 30-60 min Glu exposure. This difference is apparently determined by a dramatic increase in the susceptibility of mitochondia to Ca(2+) overload during nerve cells maturation. The exact mechanisms of Glu-induced profound MD and its coupling with the impairment of Ca(2+) extrusion following toxic Glu challenge is not clarified yet. Their elucidation demands a study of dynamic changes in local concentrations of ATP, Ca(2+), H(+), Na(+) and protein kinase C using novel methodological approaches.     

Wine polyphenols stimulate superoxide anion production to promote calcium signaling and endothelial-dependent vasodilatation

The present study was aimed to evaluate the mechanisms involved in the vasorelaxant effects of red wine polyphenol compounds (RWPC) in small mesenteric rat arteries. RWPC produce relaxation in small mesenteric arteries. This relaxant effect was abolished by endothelial denudation, NO-synthase blockade with L-NAME and partial depolarization with KCl or L-NAME plus KCl. Incubation with the reactive oxygen species scavenger, superoxide dismutase (SOD) plus catalase, or inhibition of NAD(P)H-dependent oxidoreductases with diphenyleneiodonium also inhibited RWPC induced vascular relaxation. Application of RWPC elicited a transient increase in intracellular calcium concentration ([Ca2+]i) in bovine aortic endothelial cells (BAEC), which was attenuated by a mixture of SOD and catalase. Incubation of BAEC with RWPC increased the SOD inhibitable production of O2-. These results suggest the involvement of O2- in the [Ca2+]i increase evoked by RWPC, leading to the activation of enzymes involved in the release of endothelial relaxant factors and subsequent vasodilatation of resistance arteries.     

Glutamate-induced calcium increase in myotubes depends on up-regulation of a sodium-dependent transporter

We report a study on the regulation by 2-chloro adenosine (2CA) of a glutamate (Glu) transporter in myogenic C2C12 cells. Long-term 2CA exposition significantly increased the V(max) of the Glu transporter. Moreover, 2CA-treated cells responded to Glu challenge by a rapid and transient increase in their intracellular calcium level. The above reported effects were totally abolished by treating C2C12 cells with the Na(+)-dependent Glu transporter inhibitors DL-threo-b-hydroxyaspartic acid and L-trans-pyrrolidine-2,4-dicarboxylic acid. We propose that the possible link between the Glu uptake increase and the Glu induction of calcium rise could be the depolarizing currents carried by Na(+) coupled with transporter activity.     

Induction of lactational estrus in organic piglet production

The longer lactation period required in organic piglet producing herds reduces the potential number of produced litters per sow per year compared with that of conventional production. Induction and use of lactational estrus may be a way to increase the productivity in organic production. However, if lactational estrus is to be beneficial under practical husbandry conditions, it is crucial that the majority of sows are successfully mated within a few days to make batch farrowing procedures possible. The objective of this study was to investigate the occurrence and timing of lactational estrus in an organic outdoor system based on ad libitum feeding, individual housing until Day 35 in lactation, followed by grouping and introduction of a boar and weaning of piglets after 8 wk. Five groups with four sows ((Danish Yorkshire × Danish Landrace) × Danish Duroc) in each were observed, and rank was determined by a food competition test. All sows showed lactational estrus, and 84% of these sows showed estrus within 1 wk, on average 43.5 d and 7.3 d after farrowing and boar introduction, respectively. The number of days from boar introduction to estrus increased significantly with increasing feed competition rank (the lowest number being the top rank position). Eighty-four percent of all sows were diagnosed pregnant 5 wk after estrus. Behavioral observations revealed that the average total number of copulations per estrus sow was 2.3 with a range of 0 to 5 copulations. The findings of the current study indicate that it is possible to combine lactational estrus and batch farrowing procedures to increase the number of weaned piglets per year per sow in organic piglet production based on 8 wk of lactation or more.     

Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes

We have analysed Ca²⁺ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca²⁺]_i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC₅₀ (200 nM). Upon receptor activation, [Ca²⁺]_i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca²⁺]_i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca²⁺ release is higher than other regions of the cell. Removal of extracellular Ca²⁺ or blockade of Ca²⁺ channels by inorganic cations (Cd²⁺ and Ni²⁺) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca²⁺]_i response. In the absence of Ca²⁺ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca²⁺ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca²⁺ entry is not operative. Furthermore, our experiments provide evidence for local Ca²⁺ oscillations in cells which can support both wave propagation as well as spatially discrete Ca²⁺ signalling.     

NAADP mediates ATP-induced Ca2+ signals in astrocytes

Intracellular Ca(2+) signals provide astrocytes with a specific form of excitability that enables them to regulate synaptic transmission. In this study, we demonstrate that NAADP-AM, a membrane-permeant analogue of the new second messenger nicotinic acid-adenine dinucleotide phosphate (NAADP), mobilizes Ca(2+) in astrocytes and that the response is blocked by Ned-19, an antagonist of NAADP signalling. We also show that NAADP receptors are expressed in lysosome-related acidic vesicles. Pharmacological disruption of either NAADP or lysosomal signalling reduced Ca(2+) responses induced by ATP and endothelin-1, but not by bradykinin. Furthermore, ATP increased endogenous NAADP levels. Overall, our data provide evidence for NAADP being an intracellular messenger for agonist-mediated calcium signalling in astrocytes.     

Induction of lactational estrus in organic piglet production

The longer lactation period required in organic piglet producing herds reduces the potential number of produced litters per sow per year compared with that of conventional production. Induction and use of lactational estrus may be a way to increase the productivity in organic production. However, if lactational estrus is to be beneficial under practical husbandry conditions, it is crucial that the majority of sows are successfully mated within a few days to make batch farrowing procedures possible. The objective of this study was to investigate the occurrence and timing of lactational estrus in an organic outdoor system based on ad libitum feeding, individual housing until Day 35 in lactation, followed by grouping and introduction of a boar and weaning of piglets after 8 wk. Five groups with four sows ((Danish Yorkshire × Danish Landrace) × Danish Duroc) in each were observed, and rank was determined by a food competition test. All sows showed lactational estrus, and 84% of these sows showed estrus within 1 wk, on average 43.5 d and 7.3 d after farrowing and boar introduction, respectively. The number of days from boar introduction to estrus increased significantly with increasing feed competition rank (the lowest number being the top rank position). Eighty-four percent of all sows were diagnosed pregnant 5 wk after estrus. Behavioral observations revealed that the average total number of copulations per estrus sow was 2.3 with a range of 0 to 5 copulations. The findings of the current study indicate that it is possible to combine lactational estrus and batch farrowing procedures to increase the number of weaned piglets per year per sow in organic piglet production based on 8 wk of lactation or more.     

Thyroid hormones stimulate calcium transport systems in rat intestine

Thyroid hormone status influences calcium metabolism. To elucidate the mechanism of action of thyroid hormones on transcellular transport of calcium in rat intestine, Ca(2+) influx and efflux studies were carried out in brush border membrane vesicles (BBMV) and across the basolateral membrane (BLM) of enterocytes, respectively. Steady-state uptake of Ca(2+) into BBMV as well as Ca(2+) efflux from the BLM enterocytes was significantly increased in hyperthyroid (Hyper-T) rats and decreased in hypothyroid (Hypo-T) rats as compared to euthyroid (Eu-T) rats. Kinetic studies revealed that increase in steady state Ca(2+) uptake into BBMV from hyper-T rats was fraternized with decrease in Michaelis Menten Constant (K(m)), indicating a conformational change in Ca(2+) transporter. Further, this finding was supported by significant changes in transition temperature and membrane fluidity. Increased Ca(2+) efflux across enterocytes was attributed to sodium-dependent Ca(2+) exchange activity which was significantly higher in Hyper-T rats and lower in Hypo-T rats as compared to Eu-T rats. However, there was no change in Ca(2+)-ATPase activity of BLMs of all groups. Kinetic studies of Na(+)/Ca(2+) exchanger revealed that alteration in Na(+)-dependent Ca(2+) efflux was directly associated with maximal velocity (V(max)) of exchanger among all the groups. cAMP, a potent activator of Na(+)/Ca(2+) exchanger, was found to be significantly higher in intestinal mucosa of Hyper-T rats as compared to Eu-T rats. Therefore, the results of this study suggest that Ca(2+) influx across BBM is possibly modulated by thyroid hormones by mediating changes in membrane fluidity. Thyroid hormones activated the Na(+)/Ca(2+) exchange in enterocytes possibly via cAMP-mediated pathway.     

Genetic variation in piglet mortality in outdoor organic production systems

Piglet mortality from farrowing to weaning is a major concern, especially in outdoor organic production systems. This issue might impair animal welfare and generate economic losses for the farmer. In particular, it is difficult to apply management tools that are commonly used for indoor pig production systems to organic or outdoor production systems. Genetics and breeding approaches might be used to improve piglet survival. However, knowledge remains limited on the genetic background underlying survival traits in organic pigs that are born and reared outdoors. Here, we investigated the mortality of piglets from farrowing to weaning in an outdoor organic pig population and suggested genetic strategies to reduce piglet mortality in this production system. The experiment included mortality records of piglets from farrowing to weaning (around 69 days of age). Pedigree-based threshold models were used to analyse the mortality traits of piglets at 0–3 days of age, 4–11 days, and 12 days to weaning. Stillborn piglets were included in the group of piglets that died at 0–3 days of age. We found that the mortality rate from farrowing to weaning was, on average, 19.2%. However, most piglet deaths (79.1%) occurred at 0–11 days of age. As the age of piglets increased, the direct heritability of piglet mortality rose from 0 to 0.04, whereas maternal heritability decreased from 0.03 to a non-significant value. Piglets with higher BW had a lower mortality rate. However, the genetic correlations between maternal effects on piglet mortality and piglet BW were not significant; thus, selection for piglets with higher BW at around 10 days of age, through improving maternal genetics, would not reduce piglet mortality. Piglet mortality increased from sows with increasing number of parities. Crossbreeding also reduced piglet mortality. In conclusion, selection focusing on sow genotype, the use of younger sows, and crossbreeding could contribute to maintain piglet mortality at lower levels in outdoor organic pig production systems.     

High extracellular calcium concentrations directly stimulate osteoclast apoptosis

Although the inhibitory effects of high extracellular calcium concentrations ([Ca](e)) on osteoclastic bone resorption have been known for several years, the exact mechanism remains poorly understood. The present study was performed to investigate the possible effect of [Ca](e) on osteoclast apoptosis. Using highly purified rabbit osteoclasts, we have shown that calcium directly promotes apoptosis in a dose-dependent manner which correlates with the dose range of calcium for the inhibition of bone resorption. A time-course experiment of apoptotic changes of osteoclasts cultured in presence of 1.8 or 20 mM calcium showed a significant difference after as early as 8 h of culture. After 72 h of culture, we observed that 80% of the cells cultured in the presence of 20 mM calcium displayed the typical features of apoptosis compared to only 20% in the medium containing 1.8 mM calcium. Calcium channel blockers and ryanodine abrogated the effects of [Ca](e) on apoptosis while neomycin, a calcium-sensing receptor agonist, did not alter cell viability. Taken together, these results suggest that calcium influx is involved in calcium-induced osteoclast apoptosis. Our results are consistent with the concept that in the presence of high [Ca](e) generated during bone demineralization, osteoclasts are subjected to negative-feedback regulation due, at least in part, to the induction of apoptosis.     

Fertilisation calcium signals in the ascidian egg

The egg of ascidians (urochordate), as virtually all animal and plant species, displays Ca2+ signals upon fertilisation. These Ca2+ signals are repetitive Ca2+ waves that initiate in the cortex of the egg and spread through the whole egg interior. Two series of Ca2+ waves triggered from two distinct Ca2+ wave pacemakers entrain the two meiotic divisions preceding entry into the first interphase. The second messenger inositol (1,4,5) trisphosphate (IP3) is the main mediator of these global Ca2+ waves. Other Ca2+ signalling pathways (RyR and NAADPR) are functional in the egg but they mediate localised cortical Ca2+ signals whose physiological significance remains unclear. The meiosis I Ca2+ wave pacemaker is mobile and relies on intracellular Ca2+ release from the endoplasmic reticulum (ER) induced by a large production of IP3 at the sperm aster site. The meiosis II Ca2+ wave pacemaker is stably localised in a vegetal protrusion called the contraction pole. It is probable that a local production of IP3 in the contraction pole determines the site of this second pacemaker while functional interactions between ER and mitochondria regulate its activity. Finally, a third ectopic pacemaker can be induced by a global increase in IP3, making the ascidian egg a unique system where three different Ca2+ wave pacemakers coexist in the same cell.     

Role of calcium signals in early development

The mammalian egg appears to transduce the duration, amplitude, and temporal presentation of the increase in the intracellular calcium concentration ([Ca(2+)](i)) upon fertilization. These Ca(2+) parameters have important short-term effects on the initiation and completion of early events of egg activation, as well as much later consequences for the extent of peri-implantation development. Recent studies have begun to shed light on how the egg quantitatively interprets the Ca(2+) signal (e.g., by summation of individual Ca(2+) rises) and the mechanisms by which down-stream Ca(2+) effectors, such as Ca(2+)/Calmodulin (CaM)-dependent protein kinase II (CaMKII), utilize this ionic signal to promote biological events that initiate development.     

Analysis of subcellular calcium signals in T-lymphocytes

Subcellular Ca(2+) signals were analysed in Jurkat and peripheral human T-lymphocytes by confocal Ca(2+) imaging employing an off-line deconvolution method. Stimulation of the TCR/CD3 complex in T-lymphocytes resulted in a series of subcellular pacemaker Ca(2+) signals preceding the first global Ca(2+) signal. The pacemaker signals occurred in a cytosolic "trigger" zone, which is localised close to the plasma membrane. The pacemaker signals were almost independent of extracellular Ca(2+) as shown by measurements in the absence of extracellular Ca(2+), or in the presence of the Ca(2+) channel blocker SK-F 96365. Analysis of the confocal Ca(2+) images revealed characteristic amplitudes of 82 +/- 30 to 109 +/- 21 nM, signal diameters between 2.5 +/- 0.9 and 3.5 +/- 1.5 microm and frequencies between 0.235 and 0.677 s(-1). Taken together, our data constitute the first analysis of subcellular Ca(2+) signals in T cells and indicate that the pacemaker Ca(2+) release events, which are necessary for the development of the global Ca(2+) signal, are composed of Ca(2+) release both from inositol 1,4,5-trisphosphate- and ryanodine receptors.     

Thiazides stimulate calcium absorption in urinary bladder of winter flounder

Thiazides inhibit voltage-independent NaCl absorption in the urinary bladder of the winter flounder presumably by blocking an electroneutral mucosal Na/Cl co-transporter. As thiazides stimulate calcium absorption in mammalian distal convoluted tubule while inhibiting NaCl absorption, we studied the effects of hydrochlorothiazide (HCTZ) on unidirectional 45Ca fluxes and intracellular electrical potential in short-circuited bladders to examine possible mechanisms of HCTZ effects on calcium transport. Basal secretory calcium flux was, on average, slightly larger than absorptive flux, reflecting small net calcium secretion. Mucosal addition of HCTZ (10(-4) M) stimulated absorptive calcium flux by 46% while the secretory flux was unaltered. Thus, HCTZ tended to induce net calcium absorption. Pre-treatment with serosal ouabain (10(-4) M) attenuated the HCTZ-induced increase in absorptive calcium flux. Moreover, HCTZ hyperpolarized the mucosal membrane potential by 18% as measured by conventional open-tip microelectrodes. These effects of HCTZ are consistent with the hypothesis that HCTZ indirectly stimulates Na/Ca exchange located at the serosal membrane. In conclusion, HCTZ in flounder urinary bladder, as in mammalian distal convoluted tubule, simultaneously inhibits NaCl absorption and stimulates calcium absorption. This study expands on the functional similarities between the flounder urinary bladder and the mammalian distal convoluted tubule.