The elevation of glutamate content and the amplification of insulin secretion in glucose-stimulated pancreatic islets are not causally related.
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Glucose increases insulin secretion by raising cytoplasmic Ca(2+) ([Ca(2+)](i)) in beta-cells (triggering pathway) and augmenting the efficacy of Ca(2+) on exocytosis (amplifying pathway). It has been suggested that glutamate formed from alpha-ketoglutarate is a messenger of the amplifying pathway (Maechler, P., and Wollheim, C. B. (1999) Nature 402, 685-689). This hypothesis was tested with mouse islets depolarized with 30 mm KCl (+ diazoxide) or with a saturating concentration of sulfonylurea. Because [Ca(2+)](i) was elevated under these conditions, insulin secretion was stimulated already in 0 mm glucose. The amplification of secretion produced by glucose was accompanied by an increase in islet glutamate. However, glutamine (0.5-2 mm) markedly augmented islet glutamate without affecting insulin secretion, whereas glucose augmented secretion without influencing glutamate levels when these were elevated by glutamine. Allosteric activation of glutamate dehydrogenase by BCH (2-amino 2-norbornane carboxylic acid) lowered islet glutamate but increased insulin secretion. Similar insulin secretion thus occurred at very different cellular glutamate levels. Glutamine did not affect islet [Ca(2+)](i) and pH(i), whereas glucose and BCH slightly raised pH(i) and either slightly decreased (30 mm KCl) or increased (tolbutamide) [Ca(2+)](i). The general dissociation between changes in islet glutamate and insulin secretion refutes a role of beta-cell glutamate in the amplification of insulin secretion by glucose. (+info)
The gravitropic setpoint angle of dark-grown rye seedlings and the role of ethylene.
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The orientation growth of coleoptiles of dark-grown seedlings of rye (Secale cereale L. cv. Marder II), when grown under various conditions, was analysed with respect to the gravivector ('gravitropic setpoint angle', GSA). Coleoptiles growing through moist vermiculite attain and maintain a GSA with an average of about 180 degrees, i.e. a vertical orientation. Seedlings growing uncovered either on the surface of vermiculite or positionally fixed on filter paper attain and maintain a GSA of 140-150 degrees (i.e. deviating from the vertical by an average of 30-40 degrees ). Changing the position of the embryo relative to the horizontally fixed seed kernel or of the angle of the seed with respect to gravity during germination (+/-40 degrees relative to the horizontal) had no significant effect on the subsequent GSA of both covered and uncovered seedlings. The GSA of uncovered coleoptiles could be restored close to 180 degrees by treatment of the seedlings with ethylene, either applied via ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC) as well as by fruit-released ethylene. The results are discussed with respect to the mechanism of the regulation of gravitropic growth of grass seedlings. (+info)
Ethylene perception generates gravicompetence in gravi-incompetent leaves of rye seedlings.
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The elongating leaves of young rye seedlings do not show a gravitropic response when placed horizontally. However, after treatment with ethylene, either supplied exogenously via ethephon or by application of its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), gravicompetence is seen. The inhibition of ethylene perception by 1-methylcyclopropene (MCP) prevents gravicompetence. Young rye leaves provide a useful model system in which to identify the components of the gravity sensing or response systems, the presence of which govern gravicompetence. (+info)
Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis.
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Ethylene signaling in Arabidopsis begins at a family of five ethylene receptors that regulate activity of a downstream mitogen-activated protein kinase kinase kinase, CTR1. Triple and quadruple loss-of-function ethylene receptor mutants display a constitutive ethylene response phenotype, indicating they function as negative regulators in this pathway. No ethylene-related phenotype has been described for single loss-of-function receptor mutants, although it was reported that etr1 loss-of-function mutants display a growth defect limiting plant size. In actuality, this apparent growth defect results from enhanced responsiveness to ethylene; a phenotype manifested in all tissues tested. The phenotype displayed by etr1 loss-of-function mutants was rescued by treatment with an inhibitor of ethylene perception, indicating that it is ethylene dependent. Identification of an ethylene-dependent phenotype for a loss-of-function receptor mutant gave a unique opportunity for genetic and biochemical analysis of upstream events in ethylene signaling, including demonstration that the dominant ethylene-insensitive phenotype of etr2-1 is partially dependent on ETR1. This work demonstrates that mutational loss of the ethylene receptor ETR1 alters responsiveness to ethylene in Arabidopsis and that enhanced ethylene response in Arabidopsis not only results in increased sensitivity but exaggeration of response. (+info)
Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells.
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System L is a major nutrient transport system responsible for the Na(+)-independent transport of large neutral amino acids including several essential amino acids. In malignant tumors, a system L transporter L-type amino acid transporter 1 (LAT1) is up-regulated to support tumor cell growth. LAT1 is also essential for the permeation of amino acids and amino acid-related drugs through the blood-brain barrier. To search for in vitro assay systems to examine the interaction of chemical compounds with LAT1, we have investigated the expression of system L transporters and the properties of [14C]L-leucine transport in T24 human bladder carcinoma cells. Northern blot, real-time quantitative PCR and immunofluorescence analyses have reveled that T24 cells express LAT1 in the plasma membrane together with its associating protein 4F2hc, whereas T24 cells do not express the other system L isoform LAT2. The uptake of [14C]L-leucine by T24 cells is Na(+)-independent and almost completely inhibited by system L selective inhibitor BCH. The profiles of the inhibition of [14C]L-leucine uptake by amino acids and amino acid-related compounds in T24 cells are comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [14C]L-leucine uptake is, therefore, mediated by LAT1 in T24 cells. Consistent with LAT1 in Xenopus oocytes, the efflux of preloaded [14C]L-leucine is induced by extracellularly applied substrates of LAT1 in T24 cells. This efflux measurement has been proven to be more sensitive than that in Xenopus oocytes, because triiodothyronine, thyroxine and melphalan were able to induce the efflux of preloaded [14C]L-leucine in T24 cells, which was not detected for Xenopus oocyte expression system. T24 cell is, therefore, proposed to be an excellent tool to examine the interaction of chemical compounds with LAT1. (+info)
An Arabidopsis mutant defective in jasmonate response is allelic to the auxin-signaling mutant axr1.
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A screen for Arabidopsis mutants that were insensitive to methyl jasmonate (MeJA) in an assay for seedling root growth yielded only alleles of previously isolated mutants jar1 and coi1, with one exception. Mapping of the locus and morphological characterization of the new mutant suggested it might be allelic to axr1, which had not previously been reported to show resistance to MeJA. The F(1) from a cross of the new mutant with axr1-3 did not show complementation, confirming that these are the same genes. The new allele is called axr1-24. In addition to MeJA and indole-3-acetic acid (IAA), axr1-24 had decreased sensitivity to 1-aminocyclopropane-1-carboxylic acid, 6-benzylamino-purine, epi-brassinolide, and abscisic acid. Both axr1-24 and the previously characterized axr1-3 allele were shown to be susceptible to the opportunistic pathogen Pythium irregulare, a trait found in other jasmonate response mutants, including jar1-1. The double mutant jar1-1/axr1-3 was more resistant to inhibition of root growth by MeJA and was more susceptible to P. irregulare infection than either single mutant, suggesting these genes might act in independent response pathways. In contrast, resistance to IAA in the double mutant was not different from axr1-3. Northern-blot analysis showed that IAA induced the jasmonate-responsive lipoxygenase 2, AOS, and AtVSP gene transcripts and induction was strongly impaired in axr1-3. However, transcript induction by MeJA was only minimally affected in axr1-3. This study demonstrates that in addition to auxin signaling, the AXR1 locus is involved in MeJA response, providing a mechanistic link between jasmonate and auxin-signaling pathways. (+info)
Selective blockade of drug-induced place preference conditioning by ACPC, a functional NDMA-receptor antagonist.
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ACPC (1-aminocyclopropanecarboxylic acid) is a partial agonist at the strychnine-insensitive glycine receptor site on the NMDA receptor complex, and a functional NMDA antagonist. A series of experiments was conducted to assess the effects of ACPC in a biased place conditioning paradigm. As previously reported, ACPC itself did not support either appetitive or aversive place conditioning. However, co-administration of ACPC (200 mg/kg) blocked the acquisition of place preferences conditioned using a variety of psychoactive drugs (amphetamine, cocaine, nomifensine, diazepam, morphine, nicotine). No tolerance was seen to this effect following two weeks of chronic ACPC administration. Overall, ACPC did not affect the expression of place conditioning when administered immediately before the post-conditioning test. However, these effects appeared somewhat variable between drugs, and further analysis showed that ACPC did block the expression of preferences conditioned with some drugs (diazepam, morphine, nicotine), but not others (amphetamine, cocaine, nomifensine). The effects of ACPC could not be accounted for by state dependence, as ACPC blocked morphine and cocaine place preferences when administered during both the acquisition and the expression phase of conditioning. In contrast to the blockade by ACPC of drug-induced place preferences, ACPC had no effect on the acquisition of place preferences conditioned using a variety of natural non-drug reinforcers (food, sucrose, social interaction, novelty). ACPC also had no effect on the acquisition of drug-induced place aversions (naloxone, picrotoxin). Thus, ACPC selectively blocked appetitive conditioning by drug reinforcers, without affecting either appetitive conditioning by natural reinforcers or drug-induced aversions. As place preference conditioning has been demonstrated to have high predictive validity for detecting compounds with an abuse potential in humans, this selective action suggests that ACPC might have some clinical utility in the treatment of addiction, without affecting responses to natural rewards. (+info)
Glutaminolysis and insulin secretion: from bedside to bench and back.
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Identification of regulatory mutations of glutamate dehydrogenase (GDH) in a form of congenital hyperinsulinism (GDH-HI) is providing a model for basal insulin secretion (IS) and amino acid (AA)-stimulated insulin secretion (AASIS) in which glutaminolysis plays a key role. Leucine and ADP are activators and GTP is an inhibitor of GDH. GDH-HI mutations impair GDH sensitivity to GTP inhibition, leading to fasting hypoglycemia, leucine hypersensitivity, and protein-induced hypoglycemia, indicating the importance of GDH in basal secretion and AASIS. The proposed model for glutaminolysis in IS is based on GDH providing NADH and alpha-ketoglutarate (alpha-KG) to the Krebs cycle, hence increasing the beta-cell ATP-to-ADP ratio to effect insulin release. The process operates with 1) sufficient lowering of beta-cell phosphate potential (i.e., fasting) and when 2) AAs provide leucine for allosteric activation and glutamate from transaminations. To test this hypothesis, IS studies were performed in rat and GDH-HI mouse models. In the rat study, rat islets were isolated, cultured, and then perifused in Krebs-Ringer bicarbonate buffer with 2 mmol/l glutamine using 10 mmol/l 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) or a BCH ramp after 50 or 120 min of glucose deprivation. In the GDH-HI mouse study, the H454Y GDH-HI mutation driven by the rat insulin promoter was created for H454Y beta-cell-specific expression. Cultured, isolated islets were perifused in leucine 0-10 mmol/l with 2 mmol/l glutamine 0-25 mmol/l, AA 0-10 mmol/l, or glucose 0-25 mmol/l. Rat islets displayed enhanced BCH-stimulated IS after 120 min of glucose deprivation, but not when energized by fuel. H454Y and control islets had similar glucose-stimulated IS, but H454Y mice had lower random blood glucose. Leucine-stimulated IS and AASIS occurred at lower thresholds and were greater in H454Y versus control islets. Glutamine stimulated IS in H454Y but not control islets. The clinical manifestations of GDH-HI and related animal studies suggest that GDH regulates basal IS and AASIS. Energy deprivation enhanced GDH-mediated IS, and H454Y mice were hypoglycemic, substantiating roles for GDH and its regulation by the phosphate potential in basal IS. Excessive IS from H454Y islets upon exposure to GDH substrates or stimuli indicate that regulation of GDH by the beta-cell phosphate potential plays a critical role in AASIS. These findings provide a foundation for defining pathways of basal secretion and AASIS, augmenting our understanding of beta-cell function. (+info)