Expression of Na(+)-independent amino acid transport in Xenopus laevis oocytes by injection of rabbit kidney cortex mRNA.
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Poly(A)+ mRNA was isolated from rabbit kidney cortex and injected into Xenopus laevis oocytes. Injection of mRNA resulted in a time- and dose-dependent increase in Na(+)-independent uptake of L-[3H]alanine and L-[3H]arginine. L-Alanine uptake was stimulated about 3-fold and L-arginine uptake was stimulated about 8-fold after injection of mRNA (25-50 ng, after 3-6 days) as compared with water-injected oocytes. T.I.C. of oocyte extracts suggested that the increased uptake actually represented an increase in the oocyte content of labelled L-alanine and L-arginine. The expressed L-alanine uptake, obtained by subtracting the uptake in water-injected oocytes from that in mRNA-injected oocytes, showed saturability and was inhibited completely by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and L-arginine. The expressed L-arginine uptake in mRNA-injected oocytes also showed saturability, being completely inhibited by L-dibasic amino acids) and partially inhibited by BCH. Expression of both L-alanine and L-arginine uptake showed clear cis-inhibition by cationic (e.g. L-arginine) and neutral (e.g. L-leucine) amino acids. In all, this points to the expression of a Na(+)-independent transport system with broad specificity (i.e. b degree, (+)-like). In addition, part of the expressed uptake of L-arginine could be due to a system y(+)-like transporter. After size fractionation through a sucrose density gradient, the mRNA species encoding these increased transport activities (Na(+)-independent transport of L-alanine and of L-arginine) were found in fractions of an average mRNA chain-length of 1.8-2.4 kb. On the basis of these results, we conclude that Na(+)-independent transport system(s) for L-alanine and L-arginine from rabbit renal cortical tissues, most likely proximal tubules, are expressed in Xenopus laevis oocytes. These observations may represent the first steps towards expression and cloning of these transport pathways. (+info)
Neuronal and glial gamma-aminobutyric acid+ transporters are distinct proteins.
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In the central nervous system, two subtypes of sodium- and chloride-coupled GABA transporter exist. One is sensitive to ACHC, the other to beta-alanine. They are thought to be of neuronal and glial origin, respectively. GABA transport in membrane vesicles derived from astroglial cells was found to be sodium- and chloride-dependent, electrogenic and much more sensitive to beta-alanine than to ACHC. Immunoblotting with antibodies directed against a variety of sequences of the ACHC-sensitive transporter indicated that none of these epitopes was shared by the glial transporter. (+info)
Anaplerotic input is sufficient to induce time-dependent potentiation of insulin release in rat pancreatic islets.
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Nutrients that induce biphasic insulin release, such as glucose and leucine, provide acetyl-CoA and anaplerotic input in the beta-cell. The first phase of release requires increased ATP production leading to increased intracellular Ca(2+) concentration ([Ca(2+)](i)). The second phase requires increased [Ca(2+)](i) and anaplerosis. There is strong evidence to indicate that the second phase is due to augmentation of Ca(2+)-stimulated release via the K(ATP) channel-independent pathway. To test whether the phenomenon of time-dependent potentiation (TDP) has similar properties to the ATP-sensitive K(+) channel-independent pathway, we monitored the ability of different agents that provide acetyl-CoA and anaplerotic input or both of these inputs to induce TDP. The results show that anaplerotic input is sufficient to induce TDP. Interestingly, among the agents tested, the nonsecretagogue glutamine, the nonhydrolyzable analog of leucine aminobicyclo[2.2.1]heptane-2-carboxylic acid, and succinic acid methyl ester all induced TDP, and all significantly increased alpha-ketoglutarate levels in the islets. In conclusion, anaplerosis that enhances the supply and utilization of alpha-ketoglutarate in the tricarboxylic acid cycle appears to play an essential role in the generation of TDP. (+info)
Charged residues at the 2' position of human GABAC rho 1 receptors invert ion selectivity and influence open state probability.
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The ability of members of the nicotinicoid superfamily of ligand-gated ion channels to selectively conduct anions or cations is critical to their function within the central nervous system. Recent work has demonstrated that residues at the intracellular end of the second transmembrane domain, between the -3' and 2' positions, form the ion selectivity filter of these receptors. In this study, the proline residue at the 2' position (Pro-2') at the intracellular end of the second transmembrane domain of the gamma-aminobutyric acid type C rho 1 subunit was mutated to glutamate (rho 1P2'E) and arginine (rho 1P2'R). Dilution potential experiments indicated that the charge selectivity of the rho 1P2'E receptor channels had been inverted, with the channels now becoming predominantly cation selective, indicating the ability of negatively charged residues at this 2' position to control charge selectivity. The mutation was also seen to have significantly decreased agonist potency and intrinsic efficacy. In contrast, the rho 1P2'R receptor channels were anion-selective but were now found to be constitutively open with high holding currents (inhibited by low gamma-aminobutyric acid doses and the competitive antagonist, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid alone) and increased agonist activity. Hill coefficients of both mutants were decreased, but competitive antagonist studies indicated that their binding sites were not significantly affected. (+info)
Circadian rhythms of ethylene emission in Arabidopsis.
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Ethylene controls multiple physiological processes in plants, including cell elongation. Consequently, ethylene synthesis is regulated by internal and external signals. We show that a light-entrained circadian clock regulates ethylene release from unstressed, wild-type Arabidopsis (Arabidopsis thaliana) seedlings, with a peak in the mid-subjective day. The circadian clock drives the expression of multiple ACC SYNTHASE genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. Ethylene production levels are tightly correlated with ACC SYNTHASE 8 steady-state transcript levels. The expression of this gene is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. In addition, ethylene production is controlled by the TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 genes, which are critical for all circadian rhythms yet tested in Arabidopsis. Mutation of ethylene signaling pathways did not alter the phase or period of circadian rhythms. Mutants with altered ethylene production or signaling also retained normal rhythmicity of leaf movement. We conclude that circadian rhythms of ethylene production are not critical for rhythmic growth. (+info)
Assessment of inequality of root hair density in Arabidopsis thaliana using the Gini coefficient: a close look at the effect of phosphorus and its interaction with ethylene.
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BACKGROUND AND AIMS: Root hair density (i.e. the number of root hairs per unit root length) in Arabidopsis thaliana varies among individual plants in response to different nutrient stresses. The degree of such variation, defined as inequality, serves as a unique indicator of the uniformity of response within a plant population to nutrient availability. METHODS: Using the Gini coefficient (G) as an inequality index, the inequality of root hair density in Arabidopsis thaliana 'Columbia' was evaluated under conditions of nutrient stresses; in particular the effect of phosphorus and its interaction with ethylene. KEY RESULTS: With decreasing phosphorus concentration, root hair density increased while inequality decreased logarithmically. The addition of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) under high phosphorus increased root hair density and decreased inequality by 7-fold. Inhibition of ethylene action with 1-methylcyclopropene (MCP) and silver thiosulphate (STS) under low phosphorus decreased root hair density, and increased inequality by 9-fold and 4-fold, respectively. The ethylene action inhibitors had little effect on root hair density under high phosphorus, but inequality increased 3-fold in the presence of MCP and decreased 2-fold in the presence of STS. Compared with the control, deficiencies in S, N and K increased inequality of root hair density, whereas deficiencies in P, Ca, B, Mn, Fe, Zn, Cu and Mg decreased inequality. In particular, the inequality of root hair density increased by over 2-fold under deficiencies of N or K, but decreased 14-fold under phosphorus deficiency. CONCLUSIONS: The inequality analysis indicates a strong correlation between prevalent signals from the environment (i.e. phosphorus stress) and the response of the plant, and the role of ethylene in this response. As the environmental signals become stronger, an increasing proportion of individuals respond, resulting in a decrease in variation in responsiveness among individual plants as indicated by reduced inequality. (+info)
3-O-methyl-6-18F-fluoro-L-dopa, a new tumor imaging agent: investigation of transport mechanism in vitro.
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(18)F-Labeled amino acids represent a promising class of imaging agents in tumors, particularly brain tumors. However, the determination of their potential to image peripheral tumors, possibly depending on individual transport characteristics, still remains an area of investigation. The present study investigated the transport mechanism for 3-O-methyl-6-(18)F-fluoro-L-dopa (OMFD), a novel (18)F-labeled phenylalanine derivative, into tumor cells. METHODS: OMFD has routinely and reliably been prepared for clinical use in 20%-25% radiochemical yield (decay corrected, related to (18)F-F(2)) using 6-(18)F-fluoro-L-3,4-dihydroxyphenylalanine preparation devices with minor modifications. In vitro uptake assays with HT-29 (human colon adenocarcinoma) cells, FaDu (squamous cell carcinoma) cells, and RBE4 (immortalized rat brain endothelial) cells were performed with OMFD under physiologic amino acid concentrations without and with the competitive transport inhibitors 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid and alpha-(methylamino)isobutyric acid plus serine and without or with Na(+). RESULTS: Transport inhibition experiments using specific competitive inhibitors demonstrated that uptake of OMFD in all cell lines tested was mediated mainly by the sodium-independent high-capacity amino acid transport systems. The highest OMFD uptake was in FaDu cells. CONCLUSION: OMFD seems to be a promising PET tracer for imaging of amino acid transport in tumors. (+info)
Possible involvement of CS-ACS1 and ethylene in auxin-induced peg formation of cucumber seedlings.
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BACKGROUND AND AIMS: Cucumber (Cucumis sativus) seedlings develop a peg on the concave side of the gravitropically bending transition zone between the hypocotyl and the root after seed germination. Peg initiation occurs in response to auxin when its levels in the concave side of the transition zone exceed a particular threshold through the graviresponse. Ethylene also plays an important role in peg formation, but its relationship to auxin in this event is not understood. Here, the role ethylene plays in auxin-induced peg formation is studied. METHODS: Peg formation of cucumber seedlings exposed to ethylene at different stages of growth or during exogenous auxin treatment was observed. In addition, ethylene evolution from the concave and convex sides of the transition zone was compared and their transcription of CS-ACS (1-aminocyclopropane-1-carboxylic acid synthase) genes was analysed by RT-PCR and in situ hybridization. KEY RESULTS: Seedlings treated with ethylene after peg initiation produced an enlarged peg, whereas ethylene treatment before peg initiation inhibited peg formation. Ethylene also promoted the development of the peg in the auxin-treated seedlings. Furthermore, the concave side of the transition zone at peg initiation produced more ethylene and CS-ACS1 mRNA than the convex side. CONCLUSIONS: Since CS-ACS1 is an auxin-inducible gene, the greater abundance of auxin in the concave side of the transition zone causes peg initiation and increases CS-ACS1-mediated ethylene biosynthesis, which then facilitates peg development. (+info)