Jourrial 01 rVrurochenistry. 1976. Vol. 26. pp. 83-87. Pcrgamon Press. Printed in Great Britain.
UPTAKE AND RELEASE OF NIPECOTIC ACID BY RAT BRAIN SLICES G. A. R.
JOHNSTON,
A. L. STEPHANSON and B. TWITCHIN
Department of Pharmacology, Australian National University, Canberra, ACT, Australia
(Received 19 May 1975. Accepted 28 May 1975) Abstract-Nipecotic acid, a potent inhibitor of GABA uptake, is taken up by slices of rat cerebral , can be released from these slices cortex by a sodium-dependent, 'high affinity' system (K, 11 p ~ ) and by an increased potassium ion concentration in a calcium-dependent manner. Nipecotic acid and GABA appear to be taken up by the same osmotically-sensitive structures. GABA and substances which inhibit GABA uptake also inhibit the uptake of nipecotic acid. GABA can release preloaded nipecotic acid from brain slices, and nipecolic acid can release preloaded GABA. This indicates that GABA and nipecotic acid can be counter-transported using the same mobile carrier. Nipecotic acid appears to have a higher affinity than GABA for this carrier.
CELLULAR uptake may terminate the postsynaptic action of the inhibitory transmitter GABA in the mammalian CNS (IVERSEN & NEAL,1968; CURTIS et al., 1971; KROGSGAARD-LARSEN et al., 1975) and thus inhibitors of GABA uptake have considerable pharmacological and neurochemical interest. Investigations of a variety of isoxazoles structurally related to muscimol led to the finding that nipecotic acid (piperidine-3-carboxylicacid) is a powerful non-competitive inhibitor of the uptake of GARA by slices of rat cerebral cortex (KROGSGAARD-LARSEN & JOHNSTON, 1975). This paper describes the preparation of radioactive nipecotic acid, and investigations of its uptake and release by slices of rat cerebral cortex.
Uptake of radioactive nipecotic acid arid radioactive G A B A The uptake of radioactive nipecotic acid into slices of rat cerebral cortex was studied essentially as described by IVERSEN & NEAL (1968) for the uptake of radioactive GABA. The brain slices (dimensions 0.1 x 0.1 x ca. 2 mm; 10 rng of tissue per incubation vessel) were preincubatcd for 15min at 37°C radioactive nipecotic acid added (usually 0 1 pci, final conccntration 0 . 1 6 ~ and ~ ) uptake of radioactivity by the slices measured after incubation usually for a further 10 min. Experimental details arc given in the legends to the appropriate tables and figures. The effect of varying the preincubation time during which the tissue was exppcd to the inhibitor was studied with respect to the inhibition of GABA uptake during 10 min at 25°C by nipecotic acid. The dependence of the uptake 'of radioactive nipecotic acid on the sodium ion concentration in the incubation medium was studied by replacing the usual sodium phosMATERIALS AND METHODS phate buffer with Tris-HC1 buffer (50mM, pH = 7.3) and Sources of compounds by substituting varying amounts of the sodium chloride [2.3-3H]GABA, specific activity 2 Ci/mmol, was pur- in the medium with choline chloride. chased from New England Nuclear, Boston and ['4C-carThe subcellular distribution of radioactive nipecotic acid boxyl]nicotinic acid, 61 mCi/mmol, from the Radiochemi- taken up into subcellular particles of homogenates of rat cal Centre, Amersham. cerebral cortex was also studied and compared with that & JOHNSTON, of radioactive GABA. The procedures were essentially the Nipecotic acid (KROGSGAARD-LARSEN 1 975). trans-4-aminocrotonic acid (JOHNSTONet a/., same as those described by IVERSEN & JOHNSTON (1971). 1975) and bicuculline methochloride (JOHNSTONet ul., The tissue was homogenised in 032 M-sucrose (2 m1/100 mg 1972)were prepared previously in our laboratory. All other tissue), and the nuclei and unbroken cells removed by cencompounds were purchased from commercial suppliers. trifugation (10009 for 10min). Samples (1.5 ml) of the supernatant were incubated in 15 ml of medium at 37°C Preparation of radioactive nipccotic acid for 5 min, [14C]nipecotic acid (0.25 pCi) and C3H]GABA Nipecotic acid labelled with C-14 in the carboxyl group (25pCi) added, and incubation continued for a further was prepared from ['4C-carboxyl]nicotinic acid by hydro- 10 min. After rapid cooling to 4"C, the mixtures were cengenation in the presence of a 5% rhodium on alumina trifuged at 100,000g for 20 min. The resulting pellets were catalyst as describcd previously for unlabelled nipecotic washed with cold 032 M-sucrose (5 ml) and then carefully 1963). The product was homogenous by suspended in cold sucrose (3ml). Each sample was then acid (FREIFELDER, thin-layer chromatography on plates of microcrystalline layered on to a 26ml continuous linear density gradient cellulose (Avicel) developed with n-butano1:pyridine: water (05-1.5 M-sucrose) and centrifuged at 50,000 g for 60 min (1 :1: 1, by vol), more than 98% of the radioactivity having i n a swinging bucket rotor. The tubes werc then pierced the same R , as unlabelled nipecotic acid and less than at thc bottom, fractions (each of 20 drops) collected and their radioactivity measured. 1% having the same R , as unlabelled nicotinic acid. 83
84
G. A . R .
JOHNSTON.
A. L. STEPHANSON and B. TWITCHIN
~ d c u s eof radioactive uipccotic acid
Thc rcleasc of preloaded radioactive nipecotic acid from sliccs of at cc!-chral cortcx was studicd using thc apparatus described by DAVlrs ('r ul. (1975). Thc slices (500 mg of tissue in 5ml of medium) after preincubation for 10min at 37°C were preloaded by incubation with radioactive nipecotic acid ( 0 5 pCi) for 20 min at 37°C. The slices on glass fibre filter discs (Whatman GF/A) held in filter holders (Swinnex 25, Millipore Corp.) were perfused with freshly oxygenated medium at 37°C at a rate of 0 5 ml/min. The concentrations of potassium and calcium ions in the medium were varied to assess the extent of any calciumdependent, potassium-stimulated release of radioactivity. In some experiments unlabellcd GABA was added to the medium to examine the extent of any GABA-stimulated I I release of radioactivity. The reverse experiment was also 0 5 I0 0 studied in which unlabelled nipecotic acid was added to TIM, min the medium used to perfuse slices preloaded with radioactive GABA. Results were expressed as the peak percentage increase of the stimulated release of radioactivity over the FIG. 1. Time course of the uptakc of radioactive nipecotic immediate prestimulation release (MULDER& SNYDER, acid. Slices of rat cerebral cortcx were incubated at 37°C for 15 min. [L4C]nipecotic acid (final concentration 0 1 6 1974). PM) added and uptake of radioactivity by the slices mcaMetabolism of radioactive riipecotic acid stired after furthcr incubation periods of 0. 5. 10 and 20 The extent of any metabolism of radioactive nipecotic min at 37°C. The results arc expressed in terms of tissue: acid during the course of the uptake and release exper- medium ratios calculatcd on the basis of an 80% water iments was examined by column and thin-layer chromat- content of the tissue. and are means s.1 .M. of quadruplicate experiments. ography. For analysis of radioactivity in the tissue at the end of the uptake and release experiments, the tissue slices on the filters were homogenized in 0.5 M-TCA and the homogenates centrifuged (IOOOg, 5 min). The TCA was removed from the supernatants by ether extraction, and bated with the brain slices for 15min (Table I). All the radioactivity in the aqueous fraction analysed by thin- of these inhibitors are known to inhibit the uptake layer chromatography on silica plates developed in n- of GABA in a similar manner, and none of the combutano1:acetic acid:water (8:s: 1, by vol). For analysis of pounds tested that failed to influence the uptake of radioactivity in the perfusates of the release experiments. nipecotic acid are known to influence the uptake of appropriate fractions were pooled, concentrated in uacuo, GABA under these conditions (IVERSEN & NEAL, and applied to small columns of Dowex 5 0 W x 4 (2W 1968; IVERSEN & JOHNSTON, 1971; BEARTet al., 1972). 400 mesh. H+-form). The columns were washed with The order of potency of those inhibitors structurally 0.01 M-hydrochloric acid and water, then eluted with 2 Mrelated to nipecotic acid and GABA is the same as ammonia solution. Aliquots of the washes were counted observed for GABA uptake: trans4aminocrotonic to determine radioactivity in acidic metabolites, while the alkaline eluents were evaporated to dryness in uacuo at acid > L-2,4-diaminobutyric acid > 8-aminovaleric 37 C'. resuspended in a small volume of water and analysed acid. h y thin-layer chromatography as described above. RESULTS
Tiinc course of'thr uptakr of radioactive nipecotic acid
Radioactive nipecotic acid was rapidly taken tip by slices of rat cerebral cortex attaining high tissue/medium ratios which increased linearly with respect to time up to 20min incubation at 37°C (Fig. 1). The tissue/medium ratio of approximately 30 attained after 5 min is comparable with that reported & NEAL(1968) for the uptake of radioacby IVERSEN 'tive GABA at 35°C but, unlike nipecotic acid uptake, GABA uptake does not increase linearly with respect to time after this period due to the efflux of radioactive metabolites of GABA. Inhibitors of the uptake of radioactive nipecotic acid A number of compounds were found to inhibit the uptake of radioactive nipecotic acid when preincu-
TABLE1. INHIBITORS OF THE
UPTAKE OF RADIOACTIVE NIPECOTlC ACID INTO RAT BRAIN SLICES
Inhibitor
UPTAKE AND RELEASE OF NIPECOTIC ACID BY RAT BRAIN SLICES G. A. R.
JOHNSTON,
A. L. STEPHANSON and B. TWITCHIN
Department of Pharmacology, Australian National University, Canberra, ACT, Australia
(Received 19 May 1975. Accepted 28 May 1975) Abstract-Nipecotic acid, a potent inhibitor of GABA uptake, is taken up by slices of rat cerebral , can be released from these slices cortex by a sodium-dependent, 'high affinity' system (K, 11 p ~ ) and by an increased potassium ion concentration in a calcium-dependent manner. Nipecotic acid and GABA appear to be taken up by the same osmotically-sensitive structures. GABA and substances which inhibit GABA uptake also inhibit the uptake of nipecotic acid. GABA can release preloaded nipecotic acid from brain slices, and nipecolic acid can release preloaded GABA. This indicates that GABA and nipecotic acid can be counter-transported using the same mobile carrier. Nipecotic acid appears to have a higher affinity than GABA for this carrier.
CELLULAR uptake may terminate the postsynaptic action of the inhibitory transmitter GABA in the mammalian CNS (IVERSEN & NEAL,1968; CURTIS et al., 1971; KROGSGAARD-LARSEN et al., 1975) and thus inhibitors of GABA uptake have considerable pharmacological and neurochemical interest. Investigations of a variety of isoxazoles structurally related to muscimol led to the finding that nipecotic acid (piperidine-3-carboxylicacid) is a powerful non-competitive inhibitor of the uptake of GARA by slices of rat cerebral cortex (KROGSGAARD-LARSEN & JOHNSTON, 1975). This paper describes the preparation of radioactive nipecotic acid, and investigations of its uptake and release by slices of rat cerebral cortex.
Uptake of radioactive nipecotic acid arid radioactive G A B A The uptake of radioactive nipecotic acid into slices of rat cerebral cortex was studied essentially as described by IVERSEN & NEAL (1968) for the uptake of radioactive GABA. The brain slices (dimensions 0.1 x 0.1 x ca. 2 mm; 10 rng of tissue per incubation vessel) were preincubatcd for 15min at 37°C radioactive nipecotic acid added (usually 0 1 pci, final conccntration 0 . 1 6 ~ and ~ ) uptake of radioactivity by the slices measured after incubation usually for a further 10 min. Experimental details arc given in the legends to the appropriate tables and figures. The effect of varying the preincubation time during which the tissue was exppcd to the inhibitor was studied with respect to the inhibition of GABA uptake during 10 min at 25°C by nipecotic acid. The dependence of the uptake 'of radioactive nipecotic acid on the sodium ion concentration in the incubation medium was studied by replacing the usual sodium phosMATERIALS AND METHODS phate buffer with Tris-HC1 buffer (50mM, pH = 7.3) and Sources of compounds by substituting varying amounts of the sodium chloride [2.3-3H]GABA, specific activity 2 Ci/mmol, was pur- in the medium with choline chloride. chased from New England Nuclear, Boston and ['4C-carThe subcellular distribution of radioactive nipecotic acid boxyl]nicotinic acid, 61 mCi/mmol, from the Radiochemi- taken up into subcellular particles of homogenates of rat cal Centre, Amersham. cerebral cortex was also studied and compared with that & JOHNSTON, of radioactive GABA. The procedures were essentially the Nipecotic acid (KROGSGAARD-LARSEN 1 975). trans-4-aminocrotonic acid (JOHNSTONet a/., same as those described by IVERSEN & JOHNSTON (1971). 1975) and bicuculline methochloride (JOHNSTONet ul., The tissue was homogenised in 032 M-sucrose (2 m1/100 mg 1972)were prepared previously in our laboratory. All other tissue), and the nuclei and unbroken cells removed by cencompounds were purchased from commercial suppliers. trifugation (10009 for 10min). Samples (1.5 ml) of the supernatant were incubated in 15 ml of medium at 37°C Preparation of radioactive nipccotic acid for 5 min, [14C]nipecotic acid (0.25 pCi) and C3H]GABA Nipecotic acid labelled with C-14 in the carboxyl group (25pCi) added, and incubation continued for a further was prepared from ['4C-carboxyl]nicotinic acid by hydro- 10 min. After rapid cooling to 4"C, the mixtures were cengenation in the presence of a 5% rhodium on alumina trifuged at 100,000g for 20 min. The resulting pellets were catalyst as describcd previously for unlabelled nipecotic washed with cold 032 M-sucrose (5 ml) and then carefully 1963). The product was homogenous by suspended in cold sucrose (3ml). Each sample was then acid (FREIFELDER, thin-layer chromatography on plates of microcrystalline layered on to a 26ml continuous linear density gradient cellulose (Avicel) developed with n-butano1:pyridine: water (05-1.5 M-sucrose) and centrifuged at 50,000 g for 60 min (1 :1: 1, by vol), more than 98% of the radioactivity having i n a swinging bucket rotor. The tubes werc then pierced the same R , as unlabelled nipecotic acid and less than at thc bottom, fractions (each of 20 drops) collected and their radioactivity measured. 1% having the same R , as unlabelled nicotinic acid. 83
84
G. A . R .
JOHNSTON.
A. L. STEPHANSON and B. TWITCHIN
~ d c u s eof radioactive uipccotic acid
Thc rcleasc of preloaded radioactive nipecotic acid from sliccs of at cc!-chral cortcx was studicd using thc apparatus described by DAVlrs ('r ul. (1975). Thc slices (500 mg of tissue in 5ml of medium) after preincubation for 10min at 37°C were preloaded by incubation with radioactive nipecotic acid ( 0 5 pCi) for 20 min at 37°C. The slices on glass fibre filter discs (Whatman GF/A) held in filter holders (Swinnex 25, Millipore Corp.) were perfused with freshly oxygenated medium at 37°C at a rate of 0 5 ml/min. The concentrations of potassium and calcium ions in the medium were varied to assess the extent of any calciumdependent, potassium-stimulated release of radioactivity. In some experiments unlabellcd GABA was added to the medium to examine the extent of any GABA-stimulated I I release of radioactivity. The reverse experiment was also 0 5 I0 0 studied in which unlabelled nipecotic acid was added to TIM, min the medium used to perfuse slices preloaded with radioactive GABA. Results were expressed as the peak percentage increase of the stimulated release of radioactivity over the FIG. 1. Time course of the uptakc of radioactive nipecotic immediate prestimulation release (MULDER& SNYDER, acid. Slices of rat cerebral cortcx were incubated at 37°C for 15 min. [L4C]nipecotic acid (final concentration 0 1 6 1974). PM) added and uptake of radioactivity by the slices mcaMetabolism of radioactive riipecotic acid stired after furthcr incubation periods of 0. 5. 10 and 20 The extent of any metabolism of radioactive nipecotic min at 37°C. The results arc expressed in terms of tissue: acid during the course of the uptake and release exper- medium ratios calculatcd on the basis of an 80% water iments was examined by column and thin-layer chromat- content of the tissue. and are means s.1 .M. of quadruplicate experiments. ography. For analysis of radioactivity in the tissue at the end of the uptake and release experiments, the tissue slices on the filters were homogenized in 0.5 M-TCA and the homogenates centrifuged (IOOOg, 5 min). The TCA was removed from the supernatants by ether extraction, and bated with the brain slices for 15min (Table I). All the radioactivity in the aqueous fraction analysed by thin- of these inhibitors are known to inhibit the uptake layer chromatography on silica plates developed in n- of GABA in a similar manner, and none of the combutano1:acetic acid:water (8:s: 1, by vol). For analysis of pounds tested that failed to influence the uptake of radioactivity in the perfusates of the release experiments. nipecotic acid are known to influence the uptake of appropriate fractions were pooled, concentrated in uacuo, GABA under these conditions (IVERSEN & NEAL, and applied to small columns of Dowex 5 0 W x 4 (2W 1968; IVERSEN & JOHNSTON, 1971; BEARTet al., 1972). 400 mesh. H+-form). The columns were washed with The order of potency of those inhibitors structurally 0.01 M-hydrochloric acid and water, then eluted with 2 Mrelated to nipecotic acid and GABA is the same as ammonia solution. Aliquots of the washes were counted observed for GABA uptake: trans4aminocrotonic to determine radioactivity in acidic metabolites, while the alkaline eluents were evaporated to dryness in uacuo at acid > L-2,4-diaminobutyric acid > 8-aminovaleric 37 C'. resuspended in a small volume of water and analysed acid. h y thin-layer chromatography as described above. RESULTS
Tiinc course of'thr uptakr of radioactive nipecotic acid
Radioactive nipecotic acid was rapidly taken tip by slices of rat cerebral cortex attaining high tissue/medium ratios which increased linearly with respect to time up to 20min incubation at 37°C (Fig. 1). The tissue/medium ratio of approximately 30 attained after 5 min is comparable with that reported & NEAL(1968) for the uptake of radioacby IVERSEN 'tive GABA at 35°C but, unlike nipecotic acid uptake, GABA uptake does not increase linearly with respect to time after this period due to the efflux of radioactive metabolites of GABA. Inhibitors of the uptake of radioactive nipecotic acid A number of compounds were found to inhibit the uptake of radioactive nipecotic acid when preincu-
TABLE1. INHIBITORS OF THE
UPTAKE OF RADIOACTIVE NIPECOTlC ACID INTO RAT BRAIN SLICES
Inhibitor
