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 K m for L-glutamate uptake was 146 μmol·l-1, and the maximum velocity of uptake (V max) was 12.1 pmol·mm-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 μmol·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 K m 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-cysteate≥threo-3-hydroxy-Dl-aspartate > D-aspartate≥L-aspartate> L-cysteine sulphinate > L-homocysteate≫D-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.
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Abbreviations
- ac:
-
acetate
- Ch:
-
choline
- CNS:
-
central nervous system
- cpm:
-
counts per minute
- CDTA:
-
trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acids
- HPLC:
-
high performance liquid chromatography
- K m :
-
Michaelis constant
- n app :
-
apparent number
- NMG:
-
N-methyl-D-glucamine
- Pipes:
-
Piperazine-N,N′-bis-[2-ethanesulfonic acid]
- SD:
-
standard deviation
- TEA:
-
tetraethyl-ammonium
- V :
-
velocity of uptake
- V max :
-
maximum velocity of uptake
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McLean, H., Caveney, S. Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis. J Comp Physiol B 163, 297–306 (1993). https://doi.org/10.1007/BF00347780
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DOI: https://doi.org/10.1007/BF00347780