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Pipecolic acid enhancement of GABA response in single neurons of rat brain
Neuropharmacology Vol. 25, No. 3, pp. 339-342, Printed in Great Britain. All rights reserved
PIPECOLIC
K. TAKAHAMA,
Faculty
ACID
1986 Copyright
ENHANCEMENT OF GABA RESPONSE NEURONS OF RAT BRAIN
T. HASHIMOTO. M.-W, WANG, N. AKAIKE, Y. OKANO, Y. KASE AND T. MIYATA
IN
0
0028-3908/86 $3.00 + 0.00 1986 Pergamon Press Ltd
SINGLE
T. HITOSHI.
Department of Pharmacological Sciences, of Pharmaceutical Sciences, Kumamoto University 5-l De-honmachi. Kumamoto 862. Japan (Accep&d
20 Januarry 19861
Using unit recording and microelectrophoresis, SUMMARY influence of pipecolic acid (PA), a major metabolite of in the brain, on GABA and glycine responses was lysine studied in the cerebral cortical and hippocampal pyramidal With small currents, PA had no effect on neurons of rats. the single neuron activities but enhanced GABA response The finding provides without affecting glycine response. a new evidence that PA may have a connection with central GABA system.
the next higher homologue of proline, is a major metabolite of Pipecolic acid (PA), lysine in rat brain (Chang, 1976). Biochemical (Meek, 1974; Nomura et al., 1979; Giacobini, Nomura & Schmidt-Glenewinkel, 1980; KasG et al., 1980a; Nomura, Schmidt-Glenewinkel suggest an involve& Giacobini. 1980) and pharmacological (Miyata et al., 1973) evidences ment of PA in the regulation of synaptic events in the central nervous system. Moreover, PA seems to have connections with some neurological diseases in infants such as hyperpipecolatemia (Gatfield et al., 1968). hyperlysinemia and cerebra-hepatorenal syndrome of Zellweger. Microelectrophoretic application of PA depresses in bicuculline sensitive manner unit activities of the rat brain (Ka& et al.. 198Db: Takahama et al.. 1982al. althouah the the influence'of PA on- GABA action was weaker than that of GABA. In the present study, in the rat brain was investigated since PA inhibits the cellular uptake of 3Hresponse GABA in the brain.
MATERIALS
AND
METHODS
with a-chloralose (60 Twenty-three male Wistar rats, weighing 180-230 g, anesthetized mg/kg, i.p.) plus urethane (800 mg/kg, i.p.). were used. After each animal was fixed in a The stereotaxic frame, the left fronto-parietal cerebral cortex was carefully exposed. body temperature was maintained at 37.0 f 1.0 C. For unit recording and microelectrophothe methods described in the previous paper (KasB et al., 1980b; resis. Takahama et al., 1982a) were followed. The pipettes contained the followina solutions: L-PA (0.1 M in 0.165 MNaCl solution, pH 6.0). GABA (0.005 M in 0.165 M NaCl soiution. pH 6.2). glycine (0.5 M in 0.165 M NaCl solution, pH 5.6), NaCl (0.165 M) and sucrose (0.4 M in 0.1 M NaCl soluL-PA was provided by Kyowa Hakko (Japan). Other substances were obtained from tion). commercial sources. The coordinates of sites for injection-recording and for stimulation were taken from the modification of the brain atlas of Kb;nig and Klippel (1963). Cerebral cortical unit activities were recorded from the deep pyramidal layer based on depth beneath the cortical surface and on the typical firing pattern. The method for identification of hippocampal pyramidal cells was described previously (KasC et al., 1980b).
RESULTS A representative
rate-meter
record
of the effect
339
of PA on GABA
response
in a cortical
340
Preliminary
Notes
The GABA response was enhanced by application of PA. The enneuron is shown in Fig. 1. hancement usually lasted for 2-5 min after the ejection of PA was discontinued. The enhanced action was not accompanied by change in the amplitude of unit firings. Simultaneous Low PA currents such as 5application of Na+ or sucrose did not affect the GABA response. 10 nA were usually sufficient to cause the enhancement. The magnitude of the enhancement was dependent on the intensity of PA currents. In contrast to the previous experiment PA solution was filled into the pipettes and (Kasi et al., 1980b), in which concentrated low PA currents produced the significant inhibition of the unit activities in rat brain, PA itself did not affect the basic firing rate even with the current of 15 nA. Except one cortical neuron, such an enhancement was observed on all the 23 cortical and 13 hippocampal pyramidal neurons tested. Glycine response in these neurons, however, was little affected by PA (Fig. 1).
#
10
5
10 PA
5
10
20
m
10
isi 20
5
10
20
10
20 10
20
5
10
20 30sec
Enhancing effect of PA on the inhibitory actions of GABA in a cerebral Fig. 1 cortical neuron. The enhancing effect on GABA response lasted for several minutes after PA current was turned off, whereas glycine response remained unaffected. Note that no change in basic firing rate occurred during PA application. Upper record continues on lower one.
DISCUSSION Present study demonstrated that electrophoretic PA did not affect the unit firing but enhanced the GABA response in the cortical and hippocampal pyramidal neurons. It is clear from the present result that the enhancement is neither due to current flow used for PA application nor due to local changes in osmotic pressures but due to the Ineffectiveness of PA on glycine response, supporting above description, enhancement is probably selective to GABA response.
function suggests
of that
PA. PA
Curtis et al. (1976) have reported that GABA uptake inhibitors such as nipecotic acid selectively enhanced the action of GABA but little affected actions of other inhibitory amino acids and that the enhancement is consistent with the inhibition of cellular uptake of GABA. PA is known to inhibit the cellular uptake of 3H-GABA but not 14C-glycine. 3H-NA, 3H-DA and 3H-5-HT into the brain slices (Nomura. Okuma & Seaawa, 1978: Kase et al.. 1980a). Thus, it is likely that the enhancement of GABA-response by-PA is due to the inhibition of cellular uptake of GABA. On the other hand, the effects of PA on GABA-metabolizing enzymes and on excitatory transmitter actions regain to be3clarified. Since PA facilitates high K+ -induced release of H-GABA but not H-NA and H-5-HT from the slices (Okuma, Nomura & Segawa, 1979: Kase et al.. 1980af. this action may be also involved in the en-
Preliminary
hancement
of GABA
Notes
341
response.
In the mammalian CNS, the cellular uptake of amino acids by neurons and glias fulfils 1976). Therefore, a brain constituinactivation of the amino acid transmitters (Martin, ent which coexists with a particular amino acid and affects the uptake system of the amino acid is likely to function as a modulator in neurotransmission. PA is a normal constituent (Okano et al., -1984) and has specific bindinq sites PA is released Caz+-dependently from the barin sl;ces in (Giacobini. 1983) in the brain. high K+-medium (Nomura et al., 1979: Kase et al., 1980a). In addition, there is a high af& Giacofinity uptake system for PA in the brain (Meek, 1974: Nomura, Schmidt-Grenewinkel bini, 1980; Kase' et al., 1980a). Regional distribution of PA in the brain is close to that of GABA (Miyata. Takahama & Okano. 1985). PA also potentiates the anticonvulsant activitv of phenobarbital (Takahama et al., 19826) which is postulated to be produced through modi: fication it is intriguing to presume that PA has a of central GABA systems. Therefore, physiological role as a modulator in central GABAergic transmission. On the other hand, PA needs relatively large dose to produce the effects on the uptake and release of GABA (Nomura, Okuma & Segawa. 1978; Okuma. Nomura & Segawa, 1979). though the effect is very selective (Nomura. Okuma & Segawa, 1978; Okuma. Nomura & Segawa, 1979). Further studies including the interaction of PA and GABA under unanesthesia are needed to elucidate the suggested role of PA in the brain.
ACKNOWLEDGEMENT Present work was Ministry of Education, dation, Japan.
supported in part by grant in aid for scientific research from the Science and Culture, Japan and by grant from Suzuki Memorial Foun-
REFERENCES Chang
Y.F. brain.
Curtis
(1976) Biochem.
Pipecolic biophys.
acid pathway: the major Res. Commun. 69: 174-180.
D.R., Game C.J.A. and Lodge D. (1976) inhibitory amino acids in the cat nervous and Johnston system. Rev.
Curtis D.R. nervous
G.A.R. (1974) of Physiology,
lysine
metabolic
Giacobini E., Nomura Y. thesis and metabolism
G.M. with
and Schmidt-Glenewinkel T. (1980) Pipecolic acid: in the rat brain. Cell. Molec. Biol. 25: l-9.
Kas6
Y., Takahama K., Hashimoto study of pipecolic phoretic Brain Res. 193: 608-613. and
Klippel
R.A.
(1963)
The
Rat
Brain,
D.L. (1976) Carrier-mediated transport and In: GABA in Nervous System Function (Roberts, pp. 342-368. Raven Press, New York.
and McCafferty G.P. Matthews W.D. by inhibitors of GABA uptake. J.L. Proc.
(1974) Uptake 33: 468~.
(Lajtha
orign,
Williams
of piperidine
Wilkins,
removal of GABA E., Chase, T.N.
(1979) Enhancement Pharmacologist 21:
and metabolism
and
of hippocampal 149p. and
pipecolic
M.D. and
A.,
biosyn-
K. (1980a) Pharamcoof piperidine and (in Japanese).
T., Kaisaku J., Okano Y. and Miyata T. (1980b) acid, a biogenic imino acid, in the mammalian
Martin
Meek
and Haust neuropathy
of the brain. In: Handbook of Neurochemistry, pp. 583-605. Plenum, New York.
Okano Y. and Takahama Y., Miyata T., Hirata A., Morimoto H., studies on alicyclic amines XXVI: logical uptake and release pipecolic acid in the brain slice. Foria pharmacol. jap. 76: 68p
J.F.R.
rat
Amino acid transmitters in the mammalian central Biochemistry and Pharmacology. 69: 97-188.
Kas6
Kijnig
in the
The in vivo in activation of GABA and other system. Exp. Brain Res. 25: 413-428.
Gatfield P.D., Taller R.T., Hinton G.G.. Wallace A.C., Abdelnour (1968) Hyperpipecolatemia, A new metabolic disorder associated hepatomegaly; a case study. Canad. med. Ass. J. 99: 1215-1233. Giacobini E. (1983) Imino acids Ed.), Vol. 3 (2nd edition),
route
Electrobrain.
Baltimore.
from synaptic regions. and Tower, T.N., Ed.),
recurrent
acid
in
inhibition
brain.
Fed.
Preliminary
342
Notes
Miyata T., Kamata K.. Noguchi M., Okano Y. and Kasg Y. (1973) Pharmacological Intracerebral administration of pipecolic acid. Sap. alicyclic amines XV: col. 23 Suppl.: 81p. Miyata
T., imino
and Okano Y. (1985) Endogenous levels and Takahama K. acids in mammalian brain, J. Neurochem. 44 Suppl.: S96.
of piperidine Nomura Y., Okuma Y. and Segawa T. (1978) Influence GABA and glycine into P fractions uptake of monoamines, spinal cord. J. Pharm. Dyn. 1: 251-255.
inhibitory
Schmidt-Glenewinkel T. and Giacobini E. (1980) Uptake Nomura Y., pipecolic acid by synaptosomes from mouse brain. Neurochem. Res. Y.. Kadota T.. Nagata J., Matsuda A., Ijima S., Takahama Quantification by selected ion monitoring of pipecolic acid, acid and glycine in rat brain. J. Chromatogr. 310: 251-259.
Okuma
(1979) The effect Y. Nomura Y. and Segawa T. high potassium-induced release of noradrenaline, slices. J. Pharm. Dyn, 2: 261-265.
Takahama K., Miyata T., Hashimoto T., Okano Y., A new type of cc-amino acid possessing acid: malian brain. Brain Res. 239: 294-298.
actions
of
and pipecolic acid on the of the rat brain and the
T. and Giacobini E. (1979) Nomura Y.. Okuma Y.. Segawa T., Schmidt-Glenewinkel dependent, high potassium-induced release of pipecolic acid from rat brain Neurochem. 33: 803-805.
Okano
studies on J. Pharma-
of 5:
A calciumslices. J.
piperidine 1163-1173.
and
K. and Miyata T. (1984) proline, -aminobutyric
of piperidine and pipecolic acid on serotonin and GABA from rat brain
Hitoshi T. and Kas$ bicuculline-sensitive
Takahama K., Miyata T., Okano Y., Kataoka M., Hitoshi T. and Kase of phenobarbital-induced anticonvulsant activity by pipecolic col. 81: 327-331.
Y.
(1982a) Pipecolic action in the mam-
Y. (1982b) Potentiation acid. Europ. J. Pharma-
PIPECOLIC
K. TAKAHAMA,
Faculty
ACID
1986 Copyright
ENHANCEMENT OF GABA RESPONSE NEURONS OF RAT BRAIN
T. HASHIMOTO. M.-W, WANG, N. AKAIKE, Y. OKANO, Y. KASE AND T. MIYATA
IN
0
0028-3908/86 $3.00 + 0.00 1986 Pergamon Press Ltd
SINGLE
T. HITOSHI.
Department of Pharmacological Sciences, of Pharmaceutical Sciences, Kumamoto University 5-l De-honmachi. Kumamoto 862. Japan (Accep&d
20 Januarry 19861
Using unit recording and microelectrophoresis, SUMMARY influence of pipecolic acid (PA), a major metabolite of in the brain, on GABA and glycine responses was lysine studied in the cerebral cortical and hippocampal pyramidal With small currents, PA had no effect on neurons of rats. the single neuron activities but enhanced GABA response The finding provides without affecting glycine response. a new evidence that PA may have a connection with central GABA system.
the next higher homologue of proline, is a major metabolite of Pipecolic acid (PA), lysine in rat brain (Chang, 1976). Biochemical (Meek, 1974; Nomura et al., 1979; Giacobini, Nomura & Schmidt-Glenewinkel, 1980; KasG et al., 1980a; Nomura, Schmidt-Glenewinkel suggest an involve& Giacobini. 1980) and pharmacological (Miyata et al., 1973) evidences ment of PA in the regulation of synaptic events in the central nervous system. Moreover, PA seems to have connections with some neurological diseases in infants such as hyperpipecolatemia (Gatfield et al., 1968). hyperlysinemia and cerebra-hepatorenal syndrome of Zellweger. Microelectrophoretic application of PA depresses in bicuculline sensitive manner unit activities of the rat brain (Ka& et al.. 198Db: Takahama et al.. 1982al. althouah the the influence'of PA on- GABA action was weaker than that of GABA. In the present study, in the rat brain was investigated since PA inhibits the cellular uptake of 3Hresponse GABA in the brain.
MATERIALS
AND
METHODS
with a-chloralose (60 Twenty-three male Wistar rats, weighing 180-230 g, anesthetized mg/kg, i.p.) plus urethane (800 mg/kg, i.p.). were used. After each animal was fixed in a The stereotaxic frame, the left fronto-parietal cerebral cortex was carefully exposed. body temperature was maintained at 37.0 f 1.0 C. For unit recording and microelectrophothe methods described in the previous paper (KasB et al., 1980b; resis. Takahama et al., 1982a) were followed. The pipettes contained the followina solutions: L-PA (0.1 M in 0.165 MNaCl solution, pH 6.0). GABA (0.005 M in 0.165 M NaCl soiution. pH 6.2). glycine (0.5 M in 0.165 M NaCl solution, pH 5.6), NaCl (0.165 M) and sucrose (0.4 M in 0.1 M NaCl soluL-PA was provided by Kyowa Hakko (Japan). Other substances were obtained from tion). commercial sources. The coordinates of sites for injection-recording and for stimulation were taken from the modification of the brain atlas of Kb;nig and Klippel (1963). Cerebral cortical unit activities were recorded from the deep pyramidal layer based on depth beneath the cortical surface and on the typical firing pattern. The method for identification of hippocampal pyramidal cells was described previously (KasC et al., 1980b).
RESULTS A representative
rate-meter
record
of the effect
339
of PA on GABA
response
in a cortical
340
Preliminary
Notes
The GABA response was enhanced by application of PA. The enneuron is shown in Fig. 1. hancement usually lasted for 2-5 min after the ejection of PA was discontinued. The enhanced action was not accompanied by change in the amplitude of unit firings. Simultaneous Low PA currents such as 5application of Na+ or sucrose did not affect the GABA response. 10 nA were usually sufficient to cause the enhancement. The magnitude of the enhancement was dependent on the intensity of PA currents. In contrast to the previous experiment PA solution was filled into the pipettes and (Kasi et al., 1980b), in which concentrated low PA currents produced the significant inhibition of the unit activities in rat brain, PA itself did not affect the basic firing rate even with the current of 15 nA. Except one cortical neuron, such an enhancement was observed on all the 23 cortical and 13 hippocampal pyramidal neurons tested. Glycine response in these neurons, however, was little affected by PA (Fig. 1).
#
10
5
10 PA
5
10
20
m
10
isi 20
5
10
20
10
20 10
20
5
10
20 30sec
Enhancing effect of PA on the inhibitory actions of GABA in a cerebral Fig. 1 cortical neuron. The enhancing effect on GABA response lasted for several minutes after PA current was turned off, whereas glycine response remained unaffected. Note that no change in basic firing rate occurred during PA application. Upper record continues on lower one.
DISCUSSION Present study demonstrated that electrophoretic PA did not affect the unit firing but enhanced the GABA response in the cortical and hippocampal pyramidal neurons. It is clear from the present result that the enhancement is neither due to current flow used for PA application nor due to local changes in osmotic pressures but due to the Ineffectiveness of PA on glycine response, supporting above description, enhancement is probably selective to GABA response.
function suggests
of that
PA. PA
Curtis et al. (1976) have reported that GABA uptake inhibitors such as nipecotic acid selectively enhanced the action of GABA but little affected actions of other inhibitory amino acids and that the enhancement is consistent with the inhibition of cellular uptake of GABA. PA is known to inhibit the cellular uptake of 3H-GABA but not 14C-glycine. 3H-NA, 3H-DA and 3H-5-HT into the brain slices (Nomura. Okuma & Seaawa, 1978: Kase et al.. 1980a). Thus, it is likely that the enhancement of GABA-response by-PA is due to the inhibition of cellular uptake of GABA. On the other hand, the effects of PA on GABA-metabolizing enzymes and on excitatory transmitter actions regain to be3clarified. Since PA facilitates high K+ -induced release of H-GABA but not H-NA and H-5-HT from the slices (Okuma, Nomura & Segawa, 1979: Kase et al.. 1980af. this action may be also involved in the en-
Preliminary
hancement
of GABA
Notes
341
response.
In the mammalian CNS, the cellular uptake of amino acids by neurons and glias fulfils 1976). Therefore, a brain constituinactivation of the amino acid transmitters (Martin, ent which coexists with a particular amino acid and affects the uptake system of the amino acid is likely to function as a modulator in neurotransmission. PA is a normal constituent (Okano et al., -1984) and has specific bindinq sites PA is released Caz+-dependently from the barin sl;ces in (Giacobini. 1983) in the brain. high K+-medium (Nomura et al., 1979: Kase et al., 1980a). In addition, there is a high af& Giacofinity uptake system for PA in the brain (Meek, 1974: Nomura, Schmidt-Grenewinkel bini, 1980; Kase' et al., 1980a). Regional distribution of PA in the brain is close to that of GABA (Miyata. Takahama & Okano. 1985). PA also potentiates the anticonvulsant activitv of phenobarbital (Takahama et al., 19826) which is postulated to be produced through modi: fication it is intriguing to presume that PA has a of central GABA systems. Therefore, physiological role as a modulator in central GABAergic transmission. On the other hand, PA needs relatively large dose to produce the effects on the uptake and release of GABA (Nomura, Okuma & Segawa. 1978; Okuma. Nomura & Segawa, 1979). though the effect is very selective (Nomura. Okuma & Segawa, 1978; Okuma. Nomura & Segawa, 1979). Further studies including the interaction of PA and GABA under unanesthesia are needed to elucidate the suggested role of PA in the brain.
ACKNOWLEDGEMENT Present work was Ministry of Education, dation, Japan.
supported in part by grant in aid for scientific research from the Science and Culture, Japan and by grant from Suzuki Memorial Foun-
REFERENCES Chang
Y.F. brain.
Curtis
(1976) Biochem.
Pipecolic biophys.
acid pathway: the major Res. Commun. 69: 174-180.
D.R., Game C.J.A. and Lodge D. (1976) inhibitory amino acids in the cat nervous and Johnston system. Rev.
Curtis D.R. nervous
G.A.R. (1974) of Physiology,
lysine
metabolic
Giacobini E., Nomura Y. thesis and metabolism
G.M. with
and Schmidt-Glenewinkel T. (1980) Pipecolic acid: in the rat brain. Cell. Molec. Biol. 25: l-9.
Kas6
Y., Takahama K., Hashimoto study of pipecolic phoretic Brain Res. 193: 608-613. and
Klippel
R.A.
(1963)
The
Rat
Brain,
D.L. (1976) Carrier-mediated transport and In: GABA in Nervous System Function (Roberts, pp. 342-368. Raven Press, New York.
and McCafferty G.P. Matthews W.D. by inhibitors of GABA uptake. J.L. Proc.
(1974) Uptake 33: 468~.
(Lajtha
orign,
Williams
of piperidine
Wilkins,
removal of GABA E., Chase, T.N.
(1979) Enhancement Pharmacologist 21:
and metabolism
and
of hippocampal 149p. and
pipecolic
M.D. and
A.,
biosyn-
K. (1980a) Pharamcoof piperidine and (in Japanese).
T., Kaisaku J., Okano Y. and Miyata T. (1980b) acid, a biogenic imino acid, in the mammalian
Martin
Meek
and Haust neuropathy
of the brain. In: Handbook of Neurochemistry, pp. 583-605. Plenum, New York.
Okano Y. and Takahama Y., Miyata T., Hirata A., Morimoto H., studies on alicyclic amines XXVI: logical uptake and release pipecolic acid in the brain slice. Foria pharmacol. jap. 76: 68p
J.F.R.
rat
Amino acid transmitters in the mammalian central Biochemistry and Pharmacology. 69: 97-188.
Kas6
Kijnig
in the
The in vivo in activation of GABA and other system. Exp. Brain Res. 25: 413-428.
Gatfield P.D., Taller R.T., Hinton G.G.. Wallace A.C., Abdelnour (1968) Hyperpipecolatemia, A new metabolic disorder associated hepatomegaly; a case study. Canad. med. Ass. J. 99: 1215-1233. Giacobini E. (1983) Imino acids Ed.), Vol. 3 (2nd edition),
route
Electrobrain.
Baltimore.
from synaptic regions. and Tower, T.N., Ed.),
recurrent
acid
in
inhibition
brain.
Fed.
Preliminary
342
Notes
Miyata T., Kamata K.. Noguchi M., Okano Y. and Kasg Y. (1973) Pharmacological Intracerebral administration of pipecolic acid. Sap. alicyclic amines XV: col. 23 Suppl.: 81p. Miyata
T., imino
and Okano Y. (1985) Endogenous levels and Takahama K. acids in mammalian brain, J. Neurochem. 44 Suppl.: S96.
of piperidine Nomura Y., Okuma Y. and Segawa T. (1978) Influence GABA and glycine into P fractions uptake of monoamines, spinal cord. J. Pharm. Dyn. 1: 251-255.
inhibitory
Schmidt-Glenewinkel T. and Giacobini E. (1980) Uptake Nomura Y., pipecolic acid by synaptosomes from mouse brain. Neurochem. Res. Y.. Kadota T.. Nagata J., Matsuda A., Ijima S., Takahama Quantification by selected ion monitoring of pipecolic acid, acid and glycine in rat brain. J. Chromatogr. 310: 251-259.
Okuma
(1979) The effect Y. Nomura Y. and Segawa T. high potassium-induced release of noradrenaline, slices. J. Pharm. Dyn, 2: 261-265.
Takahama K., Miyata T., Hashimoto T., Okano Y., A new type of cc-amino acid possessing acid: malian brain. Brain Res. 239: 294-298.
actions
of
and pipecolic acid on the of the rat brain and the
T. and Giacobini E. (1979) Nomura Y.. Okuma Y.. Segawa T., Schmidt-Glenewinkel dependent, high potassium-induced release of pipecolic acid from rat brain Neurochem. 33: 803-805.
Okano
studies on J. Pharma-
of 5:
A calciumslices. J.
piperidine 1163-1173.
and
K. and Miyata T. (1984) proline, -aminobutyric
of piperidine and pipecolic acid on serotonin and GABA from rat brain
Hitoshi T. and Kas$ bicuculline-sensitive
Takahama K., Miyata T., Okano Y., Kataoka M., Hitoshi T. and Kase of phenobarbital-induced anticonvulsant activity by pipecolic col. 81: 327-331.
Y.
(1982a) Pipecolic action in the mam-
Y. (1982b) Potentiation acid. Europ. J. Pharma-