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Dihydrokainic acid affects extracellular taurine and phosphoethanolamine levels in the hippocampus
J~quusc/ence Letters. 38 (1983) 67-72 Elsevier Scientif'tc Publishers Ireland Ltd.
DIHYDROKAINIC A C I D ~
67
EXTRACEIJ, ULAR TAURINE AND
PHOSPHOETHANOLAMINE LEVEL~ IN THE HIPPOCAMPUS
ANDERS LEHMANN* and ANDERS HAMBERGER
Institute of lqeurobiology and Department o f Zoophysiology, University of OOteborg, S-400 33 OOtcborg (Sweden) (Received November 291h. 1982; Revised version received Ma~ch 21st. 1983; Accepted April 7th. 1983)
Key words: dihydrokainic acid - glutamate - taurine - phnsphoethanolamine - hippocampus
The specificity of the inhibitory effect of dihydrokainic acid on the high-affinity glutamate uptake was tested in an in vivo system. The rabbit hippocampns was perfused with Krebs-Ringer bicarbonate medium to which dihydrokainic acid was added (final concentrations I and $ raM) with or without depolarizing potassium cmtcentrations. Dihydrokainate elevated the levels of glutamate, taurine and phosphoethanolamine. This response was partly similar to that observed after high potassium administration. The selectivity of dihydrokaini¢ acid is thus not restricted to glutamate, and its value as a tool in the study of glutamatergic transmission is discussed.
The discovery of the powerful excitatory and neuron-lesioning properties of kainic acid (KA) and other "excitotoxins", has focussed considerable interest on their mechanism and sites of action [3, 10, 151. The evidence for involvement of endogenous excitotoxic substances in neurological disturbances is, however, mostly circumstantial. It has been suggested, for example, that dysfunction of the corticostriatal gintamatergic tract is part of the etiology of Huntington's chorea [i4]. We have recently shown that KA, administered in vivo into the rabbit hippocamp.ts, raises extracellular glutamate levels [I I]. The mechanism of this effect co.Jld be due to at least two factors, as KA blocks the high-affinity uptake of glutamate [8] in addition to its stimulation of glutamate release [9]. The avid uptake system for glutamate is probably largely responsible for a previously observed discrepancy between the relative release of glutamate in vivo and in vitro during K+-depolarizat'~c,n [5, I I]; 2-4 times basal levels and 20-40 times basal levels, respectively. The saturated derivative of KA, dihydrokainic acid (DKA), has a biological effect different from that of KA. The convulsant [8], excitatory [7] and toxic [16] properties of KA are significantly reduced, while inhibition of the high-affinity uptake of *Author for correspondence at: Institute of Neurobiology, University of Go~eborB, P.O. Box 33 031, S-400 33 G~mborg, Sweden.
3 80
80 3
3 3 80 80
120-140 140-240
240-310 310-350 350-380 380-400
i I ! i
-
6 3 (n.s.)
34
144 ± 10 Ii0:1: 14(n,s~) 316 ± !14 230 ± 84
145 ± 94 ±
i00 218 ±
GIu
K" (raM)
DKA (raM)
Amino acids
Medium
O- 90 90-120
Time (min)
106 98 ?$ 62
± 12 (n,s,) ± 8(n,s,) ± ? ± 7
$'7 ± 14 I I I ± 13 (n.s.)
I00 65 :~ 9
Gin
342:1:202 344 ± 151 540 + 255 841 ± 484
$20 ± 3"/0 125 ~: S9 (n,s,)
I00 $17 ± 213
P-EI-NHa
128 113 728 92d
± 2"/(n,s.) ± 17(n.s.) ± 343 ± 397
913 ± 360 !00 ± 6 (n.s,)
100 686 ± 161
Tau
145 :~ 22 145 .*- 20 1"72 :t: 15 130 .*- 16
IO0 138 :t: 10 140 ± 12 99 ± 6 (n,s.)
Ala
The table presents the time course of cbanaes in amino acids in continuous perfesionexperiments, Basal levels at perfesionwith Krebs-Rinaerbicarbonate media given as 100. The dialysates were collected during the last IOmin of each time period indicated, Perfusion rate was 2,Sial/rain, Means * S,E.M. for 4 experiments. The statistical significance for the changes was calculated a~cordin8 to the Walsh test [191, All values (except for those denoted by n.s.) were significantly different from the basal level with a probability of p<0,062,
EFFECTS OF DIHYDROKAINIC ACID (DKA) ON EXTRACELLULAR CONCENTRATIONS OF ENDOGENOUS AMINO ACIDS IN THE RABBIT HIPPOCAMPUS
TABLE i
69
glutamate is the main characteristic [$]. This makes DKA a potentially useful neuropharmacological tool in the study of the transport of glutamic acid [13]. The present study was undertaken to test the selecti,dty of the uptake-inhibitory features of DKA in vivo, given alone or in combination with depolarizing concentrations of K +. The long, C into the rabbit hippocampus. The animals were allowed to recover for 24h and the
fiber was perfused with Krebs-Ringer bicarbonate (KRB) medium at a rate of 2.5 ~,i/min. The perfusate, which equilibrates with the extracellular space of the hippocampus, was collected in 10 min fractions and analyzed for endogenous amino acids in an HPLC system [12]. When a stable baseline had been obtained, the fiber was perfused with a KRB medium containing 80 mM K ÷ (an equimolar reduction of Na÷). After perfnsion with this medium for 50 rain, the normal medium was reintroduced until prestimulation levels of amino acids were obtained. DKA (1 or 5 mM) was then included in the normal medium and, I l0 min later, 80 mM K ÷ was included Jn the DKA medium. Samples were collected for amino acid analysis as indicated in Tables I and I1, The DKA was synthesized by Dr. L. BIomberg, Department of Organic Chemistry If, Univ. of Lund, Sweden, Results from model experiments indicate that the K ÷ concentration in the immediate vicinity of the fiber ~ approximately'35-40mM after 5 min 1111. Corresponding values for DKA are not available, but a total dose of 140 (1 mM DKA) and 700 (5 mM DKA) nmol had penetrated into the tissue during the perfusion as calculated according to Lehmann et al. [11]. The response to 80mM K÷ (Tables i and 11) was similar to that seen after perfusion with 56 mM K + [11], The most pronounced effects of K" were i.qereased extracellular levels of glutamate, taurine and phosphoethanolamine, in parallel, glutamine was significantly decreased. The non-neuroactive alanine increased slightly. After perfusion with DKA in the normal medium, glutamate, taurine and phosphoethanolamine increased significantly, alanine increased slightly, but glutamine was unaffected. DKA in combination with 80mM K ~ further increased the levels of glutamate, taurine and phosphoethanolamine (T~bles I and !1). A small difference was observed between the response with I and 5 mM DKA. The increase in glutamate appeared to be maximal after I mM DKA. The K+-induced decrease in glutamine was not influenced by I mM DKA, but was abolished after 5 mM DKA. A DKA concentration dependency was evident for phosphoethanolamine and taurine, the higher concentration giving a response 3 times that of the lower. DKA plus 80 mM K + did not, however, result in a similar concentration-response effect for DKA. The effect of D K A on extracellularamino acids did not differ appreciately from that of KA [11], indicating that DKA is a weak inhibitor of glutamate uptake in vivo. This is in contrast to the more potent inhibition by DKA of glutamate uptake in vitro which is reported [8]. The increase in extracellular glutamate may, however,
80 3 3 3 80 80
90-120
120-140 140-240 240-310 310-3S0 350-380 380-400
J
K* (raM)
.,,
Medium
3 80
0-90
(min)
Time
,,,,,
3 $ 5 $
-
-
,,
,,
DKA (mM)
i
i
214 219 98 149 133 330 284
I00
iJ
Glu
± ± ± ± ± ± ±
45 33 9 (n.s.) $ 12 28 20
Amino acids
,J,,
I00 76 73 120 117 114 108 134
Gin
:~ ± ± ± ± ± ±
R.,
,,
,i,
iLL
14 IS 10 (n,s.) 14(n.s.) 8 (n,s,) 12 (n,s,) 16 (n.s,)
,,
,I,,
I
108 638 ± 672 ± 130 ± 663 ± 840 ± 9S9 ± 1068 ±
I,I.
I
140 294 II (n,s,) 163 262 240 304
, I
P-Et-NH~ II
U,
,I
100 850 833 92 372 360 914 998
H,
Tau
± 112 ± 249 ± 13 (n,s,) ± 73 ± 79 ± $9 ± 48
134 I05 176 124 156 151
:i: :l: * ~ ± ±
26 13 (n,s,) 39 I0 (n,s,) 12 16
I00 12S ± I0
Ala
The table presents the time course of cbanges in amino acids in continuous perfusion experiments, Basal levels at peffusion with Krebs-Rin8er bicarbonate medium given as ~,~. The clialysates were collected durin8 the I ~ ! 10 min of each time period indicated, Perfusion rite was 2,S ~ltmin, Means ± S,E,M. for 4 experiments. The statistical si8rdficance for the changes was calculated accordin8 to the Walsh test 119], All values (except for those denoted by n,s,) were si8niflcantly different from the basal level with a probability of p<0,062.
HIPPOCAMPUS
E F F E ~ S O F DIHYDROKAINIC ACID (c,~A)O N E X T R A C E L L U L A R CONCENTRATIONS OF E N D O G E N O U S A M I N O ACIDS IN THE RABBIT
TABLE |1
71
not exclusively be due to inhibition of uptake; it may originate partly from postsynaptic neurons which are excited by DKA. Glutamine, which normally decreases extraceHularly upon K +-depolarization [11], was unaffected by K + when the hippocampus was perfnsed with 5 mM DKA simultaneously. The moderate elevation of branched chain amino acids (data not shown) could account for ~ of this, since this group o f amino acids inhibits glutamine transport [1]. As D K A did not selectively inhibit glutamate uptake, but also influenced extracellular levels of taurine and phosphoethanolamine, its usefulness for the study of glutamate uptake systems and their relevance in "'excitotoxicity" may be limited. Furthermore, the results stress the complexity of interactions which may be involved in iontophoretic-electrophysiologlcal agonist-antagonist studies: the elevation of extracellular taurine could conceivably interfere with the postsynaptic action of DKA. The physiological significance of the increase in taurine and phosphoethav.olamine and their cellular origin is presently unclear. Taurine may be important in the regulation of neuronal excitability (e.g. ref. 6). Taurine is released from several brain regions by depolarizing agents f~.,4, 17, 181. The effect on taurine in the present experiments is probably secondary to the elevated glutamate. A direct effect of DKA on taurine transport seems unlikely. The results show, furthermore, that K÷ and DKA were additive with respect to phosphoethanolamine and taurine, while a potentiation between K + and DKA seemed to occur for glutamate. A complete concentration-response graph for K ÷ and DKA was, however, not performed. A doubling of the agonist concentration may evoke a more than doubled response in the logarithmic part of the concentration-response curve (apparent potentiation). Theoretically, a potentiation indicates that two compounds t, perate in different manners to elicit a common response. In the present case, the principal effect of K + was to release glutamate from neuronal and glial cells (although inhibition of the glutamate uptake probably takes place), and the main effect of DKA may be inhibition of glutamate uptake. In summary, DKA administered in vivo augments the extracellular concentration of several amino acids and may therefore he of limited use as a specific tool in the study of glutamatergic neurotransmission. I Benjamin, A.M., Verjcc, Z.H. and Quastel, J.H., Effects of branched-chain L-amino acids, L-phenylalanine and L-methionine on the transport of L-glutamine in rat brain cortex in vitro. Influence of cations, J. Neurnchem., 35 (1980) 78-87. 2 Bernardi, N., Jurandyr, A.A., Dacke, C.G. and Davidson, N., Calciuen-dependent increase in efflux of (I-)H)-taudne from the superfuf~l rat cerebellar cortex in vivo, Pflfigers Arch., 372 (1977) 203-205. 3 Biziere, K. and Co/k, J.T., Influence of corticostdat~l afferents on striatal kainic acid neurotoxicity, Neurosci. Lett., 8 (1978) 303-310. 4 Davidson, N., High potassium, veratddinc and electrically induced release of taudne from the cerebellar cortex, J. Physiol. (Pads), 75 (1979) 673--67~. 5 Hamberger, A., Lindroth, P. and NystfOm, B., Regulation of glutamate biosynthesis and release in vitro by low levels of ammonium ions, Brain Res. 237 (1982) 339-350.
72 6 Xuxtable. R.J., ImilgMSon functiou: metabolism and pharnmcology oftaurin¢ in the brain. In The Role of Pcptid~ and Amino Acids as N c u r ~ m . Alan R. Liss Inc.. New York. 1981. pp. 53-97. 7 J ~ G,A~L. Cmxis, D~R,, ~ , L ~ McCul/och. R,M,, ~ ~ excitation I~" ~fonnat~ r e s t ~ ' ~ of ,-.gh~lic ~ Natm'e ~ ) , ~ (1978) ~ ,
sJ
on. o . A . L
S;M,E: m,d
B,
mo,,in
acid on SU-
affinity u~ake of L-glutamicacid in rat brain slices. J, Nem'ochem.. 320979) 121-127. 9 Kr~pan. B., Bed. S. and ~ . WJ.. ~ in neuronal-i~al mmabolism of ~ e by the neufotoxin kaink: acid, J. N e u t - - . . 311(19112)509-~15. I0 Kohler. C+o ~-Irwmcz. R. and Ftt~. K+. Perfogant path t ~ prmect hippocampal granule from kainat¢ lesion. Nc~rosci+ Lett., l0 (1978j 241-246. I I Lchnmmm.A.. Isacsson,H. and Hambcrgcr. A., Effects of in ,ivo administration of kainic acid on the ¢xtraceHular amino acid pool in the rabbit hippocampus. J. Neurochem., 40 (19S3) 1314-1320. 12 Lindfmh. P. and Mopper. K.. High-pcrfonu-m~ liquid d~romtograpbic determination of subpicomoh:amounts of amino acids by precolumn fluorescencederivation!ionwith o-phthaldialdehyde. Anal~. Chem.. 51 (1979) 1667-1674. 13 Lodge. D.. Johnston, G.A.R., Curtis. D.R. and Bornstcin, i.C.. Kainate neurmoxicity and gimamale inactivation. Neurosci. Left., 14 (t9791 343-348. 14 McGeer, E.O. and MclT.m~oP.L., Duplication of biochemical changes of Huminlgton's chorea by imrastriatal injections of lglutamic and kainic acids, Nature (Lond.), 263 (1976) 517-519. 15 McGeer. E.G.. McGeer. P.L. and $inlg, K., Kaitmte-induoeddegeueration of nemtriatal neurons: d~'m:l~m.T upon corticostrialal tract. Brain Res.. 139 (1978) 381-383. 16 Nadler. J.V., Eveuf~n. D.A. and Cuthlmrtson. G.J.. Comparative toxicity of kainic acid and other acidic amino acids toward rat hippo~mpal neurons. Nenroscieuc¢, 6 (1981) 2505-2517. 17 Oja, S.S., Korpi, E.R. and Kontro. P.. Potassium-stimulated release of taurme, hypolaurine and GABA from brain tissue in vitro. In F.V. D¢ Feudis and P. Mand¢l (Eds.l° Amino Acid Neurotransmiuers. Raven Pres~. New York, 1981, pp. I?~mlSI. 18 Smith, L.F.P. and Pyc~:k. C.J.. Potassium-~timulated relea~ of radiolal~elled taurine and Iglycine from the i~lated rat retina. J. Neurochcm.. 39 (19821 6.~J~658. 19 Walsh. I.E.. Applications of some si~,nificance tests for the median which are valid under very general conditions. J. Amer. 5tati.~t. Assoc., 44 (19.19) 343.
DIHYDROKAINIC A C I D ~
67
EXTRACEIJ, ULAR TAURINE AND
PHOSPHOETHANOLAMINE LEVEL~ IN THE HIPPOCAMPUS
ANDERS LEHMANN* and ANDERS HAMBERGER
Institute of lqeurobiology and Department o f Zoophysiology, University of OOteborg, S-400 33 OOtcborg (Sweden) (Received November 291h. 1982; Revised version received Ma~ch 21st. 1983; Accepted April 7th. 1983)
Key words: dihydrokainic acid - glutamate - taurine - phnsphoethanolamine - hippocampus
The specificity of the inhibitory effect of dihydrokainic acid on the high-affinity glutamate uptake was tested in an in vivo system. The rabbit hippocampns was perfused with Krebs-Ringer bicarbonate medium to which dihydrokainic acid was added (final concentrations I and $ raM) with or without depolarizing potassium cmtcentrations. Dihydrokainate elevated the levels of glutamate, taurine and phosphoethanolamine. This response was partly similar to that observed after high potassium administration. The selectivity of dihydrokaini¢ acid is thus not restricted to glutamate, and its value as a tool in the study of glutamatergic transmission is discussed.
The discovery of the powerful excitatory and neuron-lesioning properties of kainic acid (KA) and other "excitotoxins", has focussed considerable interest on their mechanism and sites of action [3, 10, 151. The evidence for involvement of endogenous excitotoxic substances in neurological disturbances is, however, mostly circumstantial. It has been suggested, for example, that dysfunction of the corticostriatal gintamatergic tract is part of the etiology of Huntington's chorea [i4]. We have recently shown that KA, administered in vivo into the rabbit hippocamp.ts, raises extracellular glutamate levels [I I]. The mechanism of this effect co.Jld be due to at least two factors, as KA blocks the high-affinity uptake of glutamate [8] in addition to its stimulation of glutamate release [9]. The avid uptake system for glutamate is probably largely responsible for a previously observed discrepancy between the relative release of glutamate in vivo and in vitro during K+-depolarizat'~c,n [5, I I]; 2-4 times basal levels and 20-40 times basal levels, respectively. The saturated derivative of KA, dihydrokainic acid (DKA), has a biological effect different from that of KA. The convulsant [8], excitatory [7] and toxic [16] properties of KA are significantly reduced, while inhibition of the high-affinity uptake of *Author for correspondence at: Institute of Neurobiology, University of Go~eborB, P.O. Box 33 031, S-400 33 G~mborg, Sweden.
3 80
80 3
3 3 80 80
120-140 140-240
240-310 310-350 350-380 380-400
i I ! i
-
6 3 (n.s.)
34
144 ± 10 Ii0:1: 14(n,s~) 316 ± !14 230 ± 84
145 ± 94 ±
i00 218 ±
GIu
K" (raM)
DKA (raM)
Amino acids
Medium
O- 90 90-120
Time (min)
106 98 ?$ 62
± 12 (n,s,) ± 8(n,s,) ± ? ± 7
$'7 ± 14 I I I ± 13 (n.s.)
I00 65 :~ 9
Gin
342:1:202 344 ± 151 540 + 255 841 ± 484
$20 ± 3"/0 125 ~: S9 (n,s,)
I00 $17 ± 213
P-EI-NHa
128 113 728 92d
± 2"/(n,s.) ± 17(n.s.) ± 343 ± 397
913 ± 360 !00 ± 6 (n.s,)
100 686 ± 161
Tau
145 :~ 22 145 .*- 20 1"72 :t: 15 130 .*- 16
IO0 138 :t: 10 140 ± 12 99 ± 6 (n,s.)
Ala
The table presents the time course of cbanaes in amino acids in continuous perfesionexperiments, Basal levels at perfesionwith Krebs-Rinaerbicarbonate media given as 100. The dialysates were collected during the last IOmin of each time period indicated, Perfusion rate was 2,Sial/rain, Means * S,E.M. for 4 experiments. The statistical significance for the changes was calculated a~cordin8 to the Walsh test [191, All values (except for those denoted by n.s.) were significantly different from the basal level with a probability of p<0,062,
EFFECTS OF DIHYDROKAINIC ACID (DKA) ON EXTRACELLULAR CONCENTRATIONS OF ENDOGENOUS AMINO ACIDS IN THE RABBIT HIPPOCAMPUS
TABLE i
69
glutamate is the main characteristic [$]. This makes DKA a potentially useful neuropharmacological tool in the study of the transport of glutamic acid [13]. The present study was undertaken to test the selecti,dty of the uptake-inhibitory features of DKA in vivo, given alone or in combination with depolarizing concentrations of K +. The long, C into the rabbit hippocampus. The animals were allowed to recover for 24h and the
fiber was perfused with Krebs-Ringer bicarbonate (KRB) medium at a rate of 2.5 ~,i/min. The perfusate, which equilibrates with the extracellular space of the hippocampus, was collected in 10 min fractions and analyzed for endogenous amino acids in an HPLC system [12]. When a stable baseline had been obtained, the fiber was perfused with a KRB medium containing 80 mM K ÷ (an equimolar reduction of Na÷). After perfnsion with this medium for 50 rain, the normal medium was reintroduced until prestimulation levels of amino acids were obtained. DKA (1 or 5 mM) was then included in the normal medium and, I l0 min later, 80 mM K ÷ was included Jn the DKA medium. Samples were collected for amino acid analysis as indicated in Tables I and I1, The DKA was synthesized by Dr. L. BIomberg, Department of Organic Chemistry If, Univ. of Lund, Sweden, Results from model experiments indicate that the K ÷ concentration in the immediate vicinity of the fiber ~ approximately'35-40mM after 5 min 1111. Corresponding values for DKA are not available, but a total dose of 140 (1 mM DKA) and 700 (5 mM DKA) nmol had penetrated into the tissue during the perfusion as calculated according to Lehmann et al. [11]. The response to 80mM K÷ (Tables i and 11) was similar to that seen after perfusion with 56 mM K + [11], The most pronounced effects of K" were i.qereased extracellular levels of glutamate, taurine and phosphoethanolamine, in parallel, glutamine was significantly decreased. The non-neuroactive alanine increased slightly. After perfusion with DKA in the normal medium, glutamate, taurine and phosphoethanolamine increased significantly, alanine increased slightly, but glutamine was unaffected. DKA in combination with 80mM K ~ further increased the levels of glutamate, taurine and phosphoethanolamine (T~bles I and !1). A small difference was observed between the response with I and 5 mM DKA. The increase in glutamate appeared to be maximal after I mM DKA. The K+-induced decrease in glutamine was not influenced by I mM DKA, but was abolished after 5 mM DKA. A DKA concentration dependency was evident for phosphoethanolamine and taurine, the higher concentration giving a response 3 times that of the lower. DKA plus 80 mM K + did not, however, result in a similar concentration-response effect for DKA. The effect of D K A on extracellularamino acids did not differ appreciately from that of KA [11], indicating that DKA is a weak inhibitor of glutamate uptake in vivo. This is in contrast to the more potent inhibition by DKA of glutamate uptake in vitro which is reported [8]. The increase in extracellular glutamate may, however,
80 3 3 3 80 80
90-120
120-140 140-240 240-310 310-3S0 350-380 380-400
J
K* (raM)
.,,
Medium
3 80
0-90
(min)
Time
,,,,,
3 $ 5 $
-
-
,,
,,
DKA (mM)
i
i
214 219 98 149 133 330 284
I00
iJ
Glu
± ± ± ± ± ± ±
45 33 9 (n.s.) $ 12 28 20
Amino acids
,J,,
I00 76 73 120 117 114 108 134
Gin
:~ ± ± ± ± ± ±
R.,
,,
,i,
iLL
14 IS 10 (n,s.) 14(n.s.) 8 (n,s,) 12 (n,s,) 16 (n.s,)
,,
,I,,
I
108 638 ± 672 ± 130 ± 663 ± 840 ± 9S9 ± 1068 ±
I,I.
I
140 294 II (n,s,) 163 262 240 304
, I
P-Et-NH~ II
U,
,I
100 850 833 92 372 360 914 998
H,
Tau
± 112 ± 249 ± 13 (n,s,) ± 73 ± 79 ± $9 ± 48
134 I05 176 124 156 151
:i: :l: * ~ ± ±
26 13 (n,s,) 39 I0 (n,s,) 12 16
I00 12S ± I0
Ala
The table presents the time course of cbanges in amino acids in continuous perfusion experiments, Basal levels at peffusion with Krebs-Rin8er bicarbonate medium given as ~,~. The clialysates were collected durin8 the I ~ ! 10 min of each time period indicated, Perfusion rite was 2,S ~ltmin, Means ± S,E,M. for 4 experiments. The statistical si8rdficance for the changes was calculated accordin8 to the Walsh test 119], All values (except for those denoted by n,s,) were si8niflcantly different from the basal level with a probability of p<0,062.
HIPPOCAMPUS
E F F E ~ S O F DIHYDROKAINIC ACID (c,~A)O N E X T R A C E L L U L A R CONCENTRATIONS OF E N D O G E N O U S A M I N O ACIDS IN THE RABBIT
TABLE |1
71
not exclusively be due to inhibition of uptake; it may originate partly from postsynaptic neurons which are excited by DKA. Glutamine, which normally decreases extraceHularly upon K +-depolarization [11], was unaffected by K + when the hippocampus was perfnsed with 5 mM DKA simultaneously. The moderate elevation of branched chain amino acids (data not shown) could account for ~ of this, since this group o f amino acids inhibits glutamine transport [1]. As D K A did not selectively inhibit glutamate uptake, but also influenced extracellular levels of taurine and phosphoethanolamine, its usefulness for the study of glutamate uptake systems and their relevance in "'excitotoxicity" may be limited. Furthermore, the results stress the complexity of interactions which may be involved in iontophoretic-electrophysiologlcal agonist-antagonist studies: the elevation of extracellular taurine could conceivably interfere with the postsynaptic action of DKA. The physiological significance of the increase in taurine and phosphoethav.olamine and their cellular origin is presently unclear. Taurine may be important in the regulation of neuronal excitability (e.g. ref. 6). Taurine is released from several brain regions by depolarizing agents f~.,4, 17, 181. The effect on taurine in the present experiments is probably secondary to the elevated glutamate. A direct effect of DKA on taurine transport seems unlikely. The results show, furthermore, that K÷ and DKA were additive with respect to phosphoethanolamine and taurine, while a potentiation between K + and DKA seemed to occur for glutamate. A complete concentration-response graph for K ÷ and DKA was, however, not performed. A doubling of the agonist concentration may evoke a more than doubled response in the logarithmic part of the concentration-response curve (apparent potentiation). Theoretically, a potentiation indicates that two compounds t, perate in different manners to elicit a common response. In the present case, the principal effect of K + was to release glutamate from neuronal and glial cells (although inhibition of the glutamate uptake probably takes place), and the main effect of DKA may be inhibition of glutamate uptake. In summary, DKA administered in vivo augments the extracellular concentration of several amino acids and may therefore he of limited use as a specific tool in the study of glutamatergic neurotransmission. I Benjamin, A.M., Verjcc, Z.H. and Quastel, J.H., Effects of branched-chain L-amino acids, L-phenylalanine and L-methionine on the transport of L-glutamine in rat brain cortex in vitro. Influence of cations, J. Neurnchem., 35 (1980) 78-87. 2 Bernardi, N., Jurandyr, A.A., Dacke, C.G. and Davidson, N., Calciuen-dependent increase in efflux of (I-)H)-taudne from the superfuf~l rat cerebellar cortex in vivo, Pflfigers Arch., 372 (1977) 203-205. 3 Biziere, K. and Co/k, J.T., Influence of corticostdat~l afferents on striatal kainic acid neurotoxicity, Neurosci. Lett., 8 (1978) 303-310. 4 Davidson, N., High potassium, veratddinc and electrically induced release of taudne from the cerebellar cortex, J. Physiol. (Pads), 75 (1979) 673--67~. 5 Hamberger, A., Lindroth, P. and NystfOm, B., Regulation of glutamate biosynthesis and release in vitro by low levels of ammonium ions, Brain Res. 237 (1982) 339-350.
72 6 Xuxtable. R.J., ImilgMSon functiou: metabolism and pharnmcology oftaurin¢ in the brain. In The Role of Pcptid~ and Amino Acids as N c u r ~ m . Alan R. Liss Inc.. New York. 1981. pp. 53-97. 7 J ~ G,A~L. Cmxis, D~R,, ~ , L ~ McCul/och. R,M,, ~ ~ excitation I~" ~fonnat~ r e s t ~ ' ~ of ,-.gh~lic ~ Natm'e ~ ) , ~ (1978) ~ ,
sJ
on. o . A . L
S;M,E: m,d
B,
mo,,in
acid on SU-
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