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Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis
Authors:H. McLean  S. Caveney
Affiliation:(1) Department of Zoology, University of Western Ontario, N6A 5B7 London, Ontario, Canada
Abstract:It is proposed that the activity of an epidermal cotransport system for Na+ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na+-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na+-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The Km for L-glutamate uptake was 146 mgrmol·l-1, and the maximum velocity of uptake (Vmax) was 12.1 pmol·mm-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mgrmol·l-1 and 8.4 pmol·mm-2·min-1. The Na+/L-glutamate co-transporter has a stoichiometry of at least two Na+ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na+ equivalent to a Km of 21 mmol · l-1 Na+. Na+ is the only external ion apparently required to drive L-glutamate uptake. Li+ substitutes weakly for Na+. Removal of external K+ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na+/K+ gradient by inhibiting epidermal Na+/K+ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteategethreo-3-hydroxy-Dl-aspartate >D-aspartategeL-aspartate>L-cysteine sulphinate >L-homocysteateGtD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na+-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na+]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,Nprime,Nprime-tetraacetic acids - HPLC high performance liquid chromatography - Km Michaelis constant - napp apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,Nprime-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - Vmax maximum velocity of uptake
Keywords:Na+/  font-variant:small-caps"  >L-glutamate co-transport  Neurotransmitter amino acids  Insect epidermis  Beetle, Tenebrio molitor
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