UPTAKE OF [14C]NIPECOTIC ACID INTO RAT DORSAL ROOT GANGLIA M. C. W. MINCHIN' Department of Pharmacology. Australian National University. Canberra. Australia (Rewired 23 O m h e r 1978. Revised 13 Drcrniber 1978. Accrptetl 3
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Ab~tract--['~C]Nipecotic acid was accumulated in isolated. desheathed rat dorsal root ganglia by a saturable process with K , , = 48.8 p~ and V,,, = 2.2 nmol/g/min. The concentration of L-2.4-diaminobutyric acid required to inhibit the uptake of nipecotic acid by 50a0 was three times the concentration of p-alanine required to d o the same. Light microscopic autoradiography indicated that the sites of uptake of ['"C]nipecotic acid were principally confined t o satellite glial cells. It is concluded that nipecotic acid is transported by the GABA uptake system in glia but that it has less affinity for this system than GABA.
NIPECOTICacid (piperidine-3-carboxylic acid) is a potent inhibitor of GABA uptake in brain slices (KROGSGAARD-LARSEN & JOHNSTON,1975), and it enhances the effects of microelectrophoretically applied GABA on the firing of cat spinal interneurones in cico (KROGSGAARD-LARSEN et a/., 1975). Furthermore. it seems that it can be counter-transported with GABA on the same mobile carrier, since GABA not only inhibits the uptake of radioactive nipecotic acid into brain slices but also stimulates its release, and vice versa (JOHNSTONet a[., 1976~).Transport processes In small brain slices are thought to be due largely to the activity of nerve terminals. although glial cells are also capable of accumulating GABA in a variety of circumstances (IVERSEN & KELLY,1975) and both processes may play an important part in removing extracellular GABA from the vicinity of the synapse after its release from inhibitory terminals. In the present study the uptake of radioactive nipecotic acid has been examined in the dorsal root ganglion. since this tissue provides a model for the investigation of GABA transport processes in glial cells (MINCHIN & IVERSEN, 1974; SCHON& KELLY,1974~1, b). MATERIALS A N D M E T H O D S ( t)Nipecotic acid labelled with carbon-14 in the carboxyl group was prepared in this laboratory, by Mr B. TWITCHIN. from [14C-carboxyl]nicotinic acid (61 mCi/ mmol, Radiochemical Centre. Amersham, U.K.) as described previously (JOHNSTON et al., 1976a). The uptake of radioactive nipecotic acid into isolated, desheathed dorsal root ganglia was studied essentially as described by SCHON& KELLY(19746) for the uptake of radioactive GABA. Single ganglia were removed from adult Wistar rats ( 2 W 3 0 0 g ) according t o the method of MI?~CHIN & IVERSEN (1974). weighed. and preincubated in 1 ml Krebs-phosphate solution (composition: 118 mM-
Present address : Department of Physiology. University of Sheffield. Sheffield S10 2TN. U.K.
NaCI. 4.8 mni-KC1, 1.2 mwCaC1,. 1.2 mw-MgSO,. 15 mMsodium phosphate buffer, pH 7.4. and 5.6 mM-n-glucose) at 25'C for 15min. [t4C]Nipecotic acid (0.1 pCi) was then added to give a final concentration usually of 1.7 p i . and the incubation was continued for 30min. At the end of the incubation the ganglia were lightly blotted. rinsed briefly four times in Krebs-phosphate solution. and dissolved overnight in 0.5 ml Soluene (Packard). The radioactivity in the ganglia was estimated after addition of 2 drops of glacial acetic acid and 10 ml 0.5",, diphenyloxazole in xylene. I n each series of experiments. blanks were included in which ganglia were incubated at 0-C for the same length of time as the experimental samples. In autoradiographic experiments ganglia were preincubated in Krebs-phosphate solution for 10 min at 37 C and then incubated in the same solution containing 7 8 ~ ~ ['4C]nipecotic acid (5 pCi;ml) for 30 min. Control ganglia were incubated in the absence of nipecotic acid. They were then rinsed briefly three times in Krebs-phosphate solution and immediately fixed. Three different fixation procedures were used; in the first the ganglia were immersed in Krebs phosphate solution containing 5",, glutaraldehyde for 1 h at room temperature, in the second the fixathe a a s IO", freshly distilled acrolein in 0.05 M-phosphate buffer pH 7.4 for 1 h at room temperature, and in the third ganglia Here frozen in n-heptane in dry ice. Following the liquid fixation procedures the ganglia were rinsed overnight at 4 C in 0.1 wphosphate buffer pH 7.4. postfixed in locl osmium tetroxide in the same buffer, rinsed, and dehydrated in 3 graded series of acetone solutions. They were then embedded in Spurr low viscosity embedding medium (Pol)sciences Inc., Warrington, PA. U.S.A.). One micron thick sections were cut, placed o n subbed microscope slides and dipped in I l h d L4 emulsion diluted I + 3 with water plus glycerol to give a final concentration of l n 0 . Following exposure at 4'C in light-tight boxes the emulsion Has developed in Kodak D K 170 for 3 min at 20 C. fixed in 25"" sodium thiosulphate for 5 min, rinsed. and mounted for light microscopic examination. Frozen ganglia were fixed t o a cryostat chuck with Ames O.C.T. compound and sectioned in the cryostat at 14pm. Sections were picked u p on subbed microscope slides and quickly dried under a hair dryer. This was accomplished in 2-5s. The slides were then dipped in 0.8",, celloidin in amyl acetate and
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FIG.1. Time course of [14C]nipecotic acid uptake. Dorsal root ganglia were incubated at 25°C with 1 . 7 p ~ - [ ' ~ C ] nipecotic acid for various times, dissolved and counted as described in Materials and Methods. Tissue-medium ratio is the ratio of the counts in 1 g of tissue to those in 1 ml of incubation medium. Each point is the mean of four determinations; vertical bars represent S.E.M. dried. Dipping and developing were carried out as described above. In experiments designed to measure the retention of radioactive nipecotic acid in the tissue following chemical fixation, ganglia were incubated with ['4C]nipecotic acid, fixed as described above, rinsed for 15 min, and dehydrated in acetone. Postfixation with osmium tetroxide was omitted since this caused unacceptable quenching. They were then digested overnight in Soluene and counted. Control ganglia were dissolved in Soluene immediately after incubation and a brief rinse. RESULTS
The ganglia slowly accumulated radioactive nipecotic acid so that after 2 h a tissue:medium ratio of 9.5 had been attained (Fig. 1). Uptake was linear for the first 30min and declined only slightly after this time. Nipecotic acid is not metabolised by brain tissue (JOHNSTON et al., 1976a), and in sensory ganglia incubated at 37°C for 30 min the uptake of [14C]nipecotic acid was not influenced by lo-' M-aminOOXyaCetiC acid (AOAA), so the slow uptake was probably not due to release of radioactive metabolites of nipecotic acid. By comparison, at 37°C the uptake of GABA into dorsal root ganglia in the presence of AOAA was much faster, tissue:medium ratios of approx 50 being attained after 2 h (SCHON & KELLY,1974b). The kinetics of ['4C]nipecotic acid uptake over a range of substrate concentrations from 1.7 to 4 0 p ~are shown in Fig. 2. The straight line double reciprocal plot is consistent with Michaelis-Menten kinetics, and the computed values of the apparent kinetic parameters were K, = 48.8 & 0 . 6 ~and~ V,,, = 2.2 f 0.4 nmol/g/min (errors are s.E.M.). In comparison,
FIG. 2. Kinetics of nipecotic acid uptake. Ganglia were incubated at 25°C for 15 min, [14C]nipecotic acid (1.7-40.0 PM) was added and the uptake of radioactivity measured following a further 30min incubation. Passive uptake was estimated from ganglia incubated with various concentrations of ['4C]nipecotic acid at OT, and subtracted from the total uptake at 25°C. V is the initial rate of uptake, S is the concentration of nipecotic acid. Each point is the mean of four determinations; vertical bars represent S.E.M.
SCHON& KELLY(1974b) found that the corresponding values for GABA uptake into dorsal root ganglia at 25°C were K, = 1 0 . 3 and ~ ~ V,,,, = 2.1 nmol/g/min. A number of substances were tested as inhibitors of nipecotic acid uptake following preincubation with the ganglia for 15 min (Table 1).GABA was the most effective of these, and p-alanine was more powerful than L-DABA. This finding was extended to the determination of the concentration giving 50% inhibition of nipecotic acid uptake (IC50) by 8-alanine and L-DABA. When tested over the range 2 x 5 x M the IC50 values were 93.0 f 22.2 PM for 8-alanine and 263.3 4 3 . 5 ~for~ L-DABA (computed values s.E.M.). These may be compared with
+
TABLE1. INHIBITORS
OF THE UPTAKE OF ['4C]NIPECOTIC
ACID BY DORSAL ROOT GANGLIA
Inhibitor (lo-"
M) ~~
GABA trans-aminocrotonic acid p-chloromercuriphenylsulphonate 8-alanine ~-2,4-diaminobutyricacid 6-aminovaleric acid
Inhibition (%)
+
84.9 2.3 69.5 f 4.5 51.6 6.1 40.3 f 2.1 20.8 & 3.3 18.4 rt 8.4
The following produced no significant inhibition at M : L-glutamate, nicotinic acid, 6-aminolevulinic acid, m-pipecolic acid, L-proline, and piperidine. Ganglia were preincubated with the inhibitor for 15 min at 25°C before the addition of [14C]nipecotic acid ( 1 . 7 ~ ~ ) . Incubation was continued for 30 min after which uptake was compared with control experiments in which no inhibitor was present. Values are mean & S.E.M.of four experiments.
FIG.3. a, b. Dark field and phase contrast micrographs of dorsal root ganglia incubated with ['4C]nipecotic acid, frozen, and prepared as described in Materials and Methods. Phase contrast of the unstained frozen section is poor but neurones, two of which are outlined by arrowheads, are identifiable. The surface of the ganglion runs along the top of the field while sensory fibres can be seen in the bottom left hand corner. The lines of high contrast surrounding neurone clusters are artefacts. The dark field micrograph shows silver grains principally over satellite glial cells lying between the neurones, with slight labelling of the sensory fibres. Exposure time 14 weeks. c, d. Dark and bright field micrographs of dorsal root ganglion incubated with [I4C]nipecotic acid, fixed with acrolein, postfixed with OsO,, dehydrated, and embedded in Spurr medium. Bright field shows neurones (large arrows) surrounded by satellite glia (small arrows) with part of the sensory fibre tract on the left. The dark field micrograph shows preferential labelling of the glia, although there is a moderate grain density over neurones and sensory fibres some of which is due to background. Exposure time 8 months. Calibration bars = 50 pm.
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Nipecotic acid uptake into glia the IC,, values of 120,uM and 7 0 0 , ~found ~ for fl-alanine and L-DABA respectively against GABA uptake in sensory ganglia by SCHON& KELLY(1974b). Autoradiographs of frozen ganglia incubated with [‘4C]nipecotic acid showed that the radioactivity was associated mostly with the satellite glial cells, but silver grains were also found over the sensory fibres and to a small extent over the neurones (Fig. 3a,b). Control ganglia processed for autoradiography and exposed for the same time as the radioactive sections showed no positive chemography ; however control sections on which the emulsion had been fogged showed occasional patches where the silver grain density was low. This may have been due to negative chemography, despite the celloidin layer, or to thinning of the emulsion over humps in the sections; the patches occurred indiscriminately over the tissue, which would argue against selective negative chemography. This pattern was only seen infrequently over the radioactive sections, and therefore the distribution of grains seen in Fig. 3 is considered to represent the true distribution of carbon-14 in the tissue. The retention of [14C]nipecotic acid by the ganglia following fixation with either glutaraldehyde or acrolein was very poor, being only 6”/, and 12%, respectively. These levels are, of course, unacceptably low for autoradiographic purposes since there is a possibility that retained material may be preferentially localised in a way unrelated to the original distribution of radioactivity in the tissue. Nevertheless, autoradiographs were obtained following acrolein fixation after a very long exposure, and are presented here since they compare favourably with those obtained from frozen ganglia (Fig. 3c,d). Again, silver grains were seen principally over satellite glia, while the sensory fibres and neurones appeared to be labelled to a similar extent. Tests for positive and negative chemography showed that neither had occurred. Some autoradiographs were obtained from ganglia incubated at 25°C and thcse showed a similar distribution of radioactivity to that found at 37°C. DISCUSSION
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potency of the other inhibitors of the uptake of nipecotic acid into sensory ganglia is similar to that for nipecotic acid uptake into brain slices, and none of the compounds which failed to inhibit nipecotic acid uptake into the ganglia influenced its uptake into et al., 1976~). brain slices, and vice versa (JOHNSTON One exception to this was p-mercuriphenylsulphonate which was less effective as an inhibitor of nipecotic acid uptake into sensory ganglia than into brain slices, as was found for GABA uptake into these two tissues (IVERSEN & JOHNSTON, 1971; JOHNSTON et al., 1976a; SCHON& KELLY,1974b). It appears therefore that nipecotic acid is transported by the same glial system that mediates GABA uptake into sensory ganglia, and the light microscope autoradiographs presented here would lend some support to this. However, the labelling of fibre tracts and neurones was greater following [‘‘C]nipecotic acid uptake than after C3H]GABA uptake into the ganglia (SCHON& KELLY,1974~).This may be due to the lower affinity of the nipecotic acid uptake process, since the difference between the localisation of the high affinity and the less specific, low affinity uptake process would become less marked. As SCHON & KELLY(1974b) have shown, the distribution of radioactivity following low affinity uptake of C3H]GABA into sensory ganglia is similar to that found in this study for nipecotic acid. It is interesting to note that while nipecotic acid is transported by the GABA system in both neurones and glia it has less affinity than GABA for the glial system and a greater affinity than GABA for the neuronal system. Moreover (-)-nipecotic acid, which has an affinity for the neuronal GABA carrier approx 5 times higher than the (+) isomer, has a configuration remarkably similar to that of (+)-DABA and like the latter shows a marked selectivity towards the neuronal GABA uptake system (JOHNSTONet ul., 1976b). BOWERYet a[. (1976) have similarly shown that (-k) nipecotic acid is more effective as an inhibitor of GABA uptake into cortical slices (‘neuronal uptake’) than into superior cervical ganglia (‘glial uptake’). It may be inferred therefore that the folded conformation and interionic distance of nipecotic acid (JOHNSTON et ul., 1 9 7 6 ~ are ) complementary to the carrier site in nerve endings, while the carrier site on glial cells has slightly different requirements. Since p-alanine is an inhibitor of both GABA and nipecotic acid uptake into glia and has a charge separation which is similar to that of nipecotic acid (JOHNSTON et al., 1976u), it appears that the charge separation of nipecotic acid is satisfactory but that its conformation does not match so well the configuration of the glial GABA carrier site.
The uptake of [‘4C]nipecotic acid displays similar saturable kinetics to the uptake of C3H]GABA into dorsal root ganglia, although it does seem to have a lower affinity constant (SCHON& KELLY, 1974b). This may in part explain the lower tissue:medium ratios achieved by nipecotic acid, although the comparative figures for GABA were obtained at a higher temperature (SCHON& KELLY,1974b). In addition, the similarity between nipecotic acid and GABA uptake is shown by the fact that in sensory ganglia fl-alanine is considerably more effective as an inhibitor of both uptake systems than is REFERENCES L-DABA. In small brain slices L-DABA is a more powerful inhibitor of the uptake of both nipecotic BOWERYN. G., JONES G. P.& NEALM. J. (1976) Selective acid and GABA than is P-alanine (IVERSEN& JOHNinhibition of neuronal GABA uptake by cis-1,3-aminocyclohexane carboxylic acid. Nature, Lond. 264, 281-284. STON, 1971; JOHNSTONet al., 1976~).The order of
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IVERSEN L. L. & JOHNSTON G. A. R. (1971) GABA uptake acid, various isoxazoles and related compounds. J . in rat central nervous system: comparison of uptake in Neurochem. 25, 797-802. slices and homogenates and the effects of some inhibi- KROGSGAARD-LARSEN P., JOHNSTON G. A. R., CURTISD. R., tors. J. Neurochern. 18, 1939-1950. GAMEC. J. A. & MCCULLOCH R . M. (1975) Structure IVERSENL. L. & KELLYJ. S. (1975) Uptake and metabolism and biological activity of a series of conformationally of y-aminobutyric acid by neurones and glial cells. Biorestricted analogues of GABA. J . Neurochem. 25, 80-V chem. Pharmac. 24, 933-938. 809. JOHNSTON G . A. R.,STEPHANSON A. I>. & TWITCHIN B. MINCHINM. C. W. & IVERSEN L. L. (1974) Release of (19760) Uptake and release of nipecotic acid by rat brain [3H]gamma-aminobutyric acid from glial cells in rat slices. J. Neurochem. 26, 83-87. dorsal root ganglia. J . Neurochem. 23, 533-540. JOHNSTON G . A. R., KROGSGAARD-LARSEN P., STEPHANSONSCHON F. & KELLY J. S. (19740) Autoradiographic localisaA. L. & TWITCHIN B. (19766) Inhibition of the uptake tion of 13H]GABA and [3H]glutamate over satellite glial cells. Brain Res. 66, 275-288. of GABA and related amino acids in rat brain slices by the optical isomers of nipecotic acid. J . Neurochem. SCHONF. & KELLYJ. S. (1974b) The characterisation of 26, 1029-1032. ['HIGABA uptake into the satellite glial cells of rat KROGSGAARD-LARSEN P. &JOHNSTON G. A. R. (1975) Inhisensory ganglia. Brain Res. 66, 289-300. bition of GABA uptake in rat brain slices by nipecotic
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Ab~tract--['~C]Nipecotic acid was accumulated in isolated. desheathed rat dorsal root ganglia by a saturable process with K , , = 48.8 p~ and V,,, = 2.2 nmol/g/min. The concentration of L-2.4-diaminobutyric acid required to inhibit the uptake of nipecotic acid by 50a0 was three times the concentration of p-alanine required to d o the same. Light microscopic autoradiography indicated that the sites of uptake of ['"C]nipecotic acid were principally confined t o satellite glial cells. It is concluded that nipecotic acid is transported by the GABA uptake system in glia but that it has less affinity for this system than GABA.
NIPECOTICacid (piperidine-3-carboxylic acid) is a potent inhibitor of GABA uptake in brain slices (KROGSGAARD-LARSEN & JOHNSTON,1975), and it enhances the effects of microelectrophoretically applied GABA on the firing of cat spinal interneurones in cico (KROGSGAARD-LARSEN et a/., 1975). Furthermore. it seems that it can be counter-transported with GABA on the same mobile carrier, since GABA not only inhibits the uptake of radioactive nipecotic acid into brain slices but also stimulates its release, and vice versa (JOHNSTONet a[., 1976~).Transport processes In small brain slices are thought to be due largely to the activity of nerve terminals. although glial cells are also capable of accumulating GABA in a variety of circumstances (IVERSEN & KELLY,1975) and both processes may play an important part in removing extracellular GABA from the vicinity of the synapse after its release from inhibitory terminals. In the present study the uptake of radioactive nipecotic acid has been examined in the dorsal root ganglion. since this tissue provides a model for the investigation of GABA transport processes in glial cells (MINCHIN & IVERSEN, 1974; SCHON& KELLY,1974~1, b). MATERIALS A N D M E T H O D S ( t)Nipecotic acid labelled with carbon-14 in the carboxyl group was prepared in this laboratory, by Mr B. TWITCHIN. from [14C-carboxyl]nicotinic acid (61 mCi/ mmol, Radiochemical Centre. Amersham, U.K.) as described previously (JOHNSTON et al., 1976a). The uptake of radioactive nipecotic acid into isolated, desheathed dorsal root ganglia was studied essentially as described by SCHON& KELLY(19746) for the uptake of radioactive GABA. Single ganglia were removed from adult Wistar rats ( 2 W 3 0 0 g ) according t o the method of MI?~CHIN & IVERSEN (1974). weighed. and preincubated in 1 ml Krebs-phosphate solution (composition: 118 mM-
Present address : Department of Physiology. University of Sheffield. Sheffield S10 2TN. U.K.
NaCI. 4.8 mni-KC1, 1.2 mwCaC1,. 1.2 mw-MgSO,. 15 mMsodium phosphate buffer, pH 7.4. and 5.6 mM-n-glucose) at 25'C for 15min. [t4C]Nipecotic acid (0.1 pCi) was then added to give a final concentration usually of 1.7 p i . and the incubation was continued for 30min. At the end of the incubation the ganglia were lightly blotted. rinsed briefly four times in Krebs-phosphate solution. and dissolved overnight in 0.5 ml Soluene (Packard). The radioactivity in the ganglia was estimated after addition of 2 drops of glacial acetic acid and 10 ml 0.5",, diphenyloxazole in xylene. I n each series of experiments. blanks were included in which ganglia were incubated at 0-C for the same length of time as the experimental samples. In autoradiographic experiments ganglia were preincubated in Krebs-phosphate solution for 10 min at 37 C and then incubated in the same solution containing 7 8 ~ ~ ['4C]nipecotic acid (5 pCi;ml) for 30 min. Control ganglia were incubated in the absence of nipecotic acid. They were then rinsed briefly three times in Krebs-phosphate solution and immediately fixed. Three different fixation procedures were used; in the first the ganglia were immersed in Krebs phosphate solution containing 5",, glutaraldehyde for 1 h at room temperature, in the second the fixathe a a s IO", freshly distilled acrolein in 0.05 M-phosphate buffer pH 7.4 for 1 h at room temperature, and in the third ganglia Here frozen in n-heptane in dry ice. Following the liquid fixation procedures the ganglia were rinsed overnight at 4 C in 0.1 wphosphate buffer pH 7.4. postfixed in locl osmium tetroxide in the same buffer, rinsed, and dehydrated in 3 graded series of acetone solutions. They were then embedded in Spurr low viscosity embedding medium (Pol)sciences Inc., Warrington, PA. U.S.A.). One micron thick sections were cut, placed o n subbed microscope slides and dipped in I l h d L4 emulsion diluted I + 3 with water plus glycerol to give a final concentration of l n 0 . Following exposure at 4'C in light-tight boxes the emulsion Has developed in Kodak D K 170 for 3 min at 20 C. fixed in 25"" sodium thiosulphate for 5 min, rinsed. and mounted for light microscopic examination. Frozen ganglia were fixed t o a cryostat chuck with Ames O.C.T. compound and sectioned in the cryostat at 14pm. Sections were picked u p on subbed microscope slides and quickly dried under a hair dryer. This was accomplished in 2-5s. The slides were then dipped in 0.8",, celloidin in amyl acetate and
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FIG.1. Time course of [14C]nipecotic acid uptake. Dorsal root ganglia were incubated at 25°C with 1 . 7 p ~ - [ ' ~ C ] nipecotic acid for various times, dissolved and counted as described in Materials and Methods. Tissue-medium ratio is the ratio of the counts in 1 g of tissue to those in 1 ml of incubation medium. Each point is the mean of four determinations; vertical bars represent S.E.M. dried. Dipping and developing were carried out as described above. In experiments designed to measure the retention of radioactive nipecotic acid in the tissue following chemical fixation, ganglia were incubated with ['4C]nipecotic acid, fixed as described above, rinsed for 15 min, and dehydrated in acetone. Postfixation with osmium tetroxide was omitted since this caused unacceptable quenching. They were then digested overnight in Soluene and counted. Control ganglia were dissolved in Soluene immediately after incubation and a brief rinse. RESULTS
The ganglia slowly accumulated radioactive nipecotic acid so that after 2 h a tissue:medium ratio of 9.5 had been attained (Fig. 1). Uptake was linear for the first 30min and declined only slightly after this time. Nipecotic acid is not metabolised by brain tissue (JOHNSTON et al., 1976a), and in sensory ganglia incubated at 37°C for 30 min the uptake of [14C]nipecotic acid was not influenced by lo-' M-aminOOXyaCetiC acid (AOAA), so the slow uptake was probably not due to release of radioactive metabolites of nipecotic acid. By comparison, at 37°C the uptake of GABA into dorsal root ganglia in the presence of AOAA was much faster, tissue:medium ratios of approx 50 being attained after 2 h (SCHON & KELLY,1974b). The kinetics of ['4C]nipecotic acid uptake over a range of substrate concentrations from 1.7 to 4 0 p ~are shown in Fig. 2. The straight line double reciprocal plot is consistent with Michaelis-Menten kinetics, and the computed values of the apparent kinetic parameters were K, = 48.8 & 0 . 6 ~and~ V,,, = 2.2 f 0.4 nmol/g/min (errors are s.E.M.). In comparison,
FIG. 2. Kinetics of nipecotic acid uptake. Ganglia were incubated at 25°C for 15 min, [14C]nipecotic acid (1.7-40.0 PM) was added and the uptake of radioactivity measured following a further 30min incubation. Passive uptake was estimated from ganglia incubated with various concentrations of ['4C]nipecotic acid at OT, and subtracted from the total uptake at 25°C. V is the initial rate of uptake, S is the concentration of nipecotic acid. Each point is the mean of four determinations; vertical bars represent S.E.M.
SCHON& KELLY(1974b) found that the corresponding values for GABA uptake into dorsal root ganglia at 25°C were K, = 1 0 . 3 and ~ ~ V,,,, = 2.1 nmol/g/min. A number of substances were tested as inhibitors of nipecotic acid uptake following preincubation with the ganglia for 15 min (Table 1).GABA was the most effective of these, and p-alanine was more powerful than L-DABA. This finding was extended to the determination of the concentration giving 50% inhibition of nipecotic acid uptake (IC50) by 8-alanine and L-DABA. When tested over the range 2 x 5 x M the IC50 values were 93.0 f 22.2 PM for 8-alanine and 263.3 4 3 . 5 ~for~ L-DABA (computed values s.E.M.). These may be compared with
+
TABLE1. INHIBITORS
OF THE UPTAKE OF ['4C]NIPECOTIC
ACID BY DORSAL ROOT GANGLIA
Inhibitor (lo-"
M) ~~
GABA trans-aminocrotonic acid p-chloromercuriphenylsulphonate 8-alanine ~-2,4-diaminobutyricacid 6-aminovaleric acid
Inhibition (%)
+
84.9 2.3 69.5 f 4.5 51.6 6.1 40.3 f 2.1 20.8 & 3.3 18.4 rt 8.4
The following produced no significant inhibition at M : L-glutamate, nicotinic acid, 6-aminolevulinic acid, m-pipecolic acid, L-proline, and piperidine. Ganglia were preincubated with the inhibitor for 15 min at 25°C before the addition of [14C]nipecotic acid ( 1 . 7 ~ ~ ) . Incubation was continued for 30 min after which uptake was compared with control experiments in which no inhibitor was present. Values are mean & S.E.M.of four experiments.
FIG.3. a, b. Dark field and phase contrast micrographs of dorsal root ganglia incubated with ['4C]nipecotic acid, frozen, and prepared as described in Materials and Methods. Phase contrast of the unstained frozen section is poor but neurones, two of which are outlined by arrowheads, are identifiable. The surface of the ganglion runs along the top of the field while sensory fibres can be seen in the bottom left hand corner. The lines of high contrast surrounding neurone clusters are artefacts. The dark field micrograph shows silver grains principally over satellite glial cells lying between the neurones, with slight labelling of the sensory fibres. Exposure time 14 weeks. c, d. Dark and bright field micrographs of dorsal root ganglion incubated with [I4C]nipecotic acid, fixed with acrolein, postfixed with OsO,, dehydrated, and embedded in Spurr medium. Bright field shows neurones (large arrows) surrounded by satellite glia (small arrows) with part of the sensory fibre tract on the left. The dark field micrograph shows preferential labelling of the glia, although there is a moderate grain density over neurones and sensory fibres some of which is due to background. Exposure time 8 months. Calibration bars = 50 pm.
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Nipecotic acid uptake into glia the IC,, values of 120,uM and 7 0 0 , ~found ~ for fl-alanine and L-DABA respectively against GABA uptake in sensory ganglia by SCHON& KELLY(1974b). Autoradiographs of frozen ganglia incubated with [‘4C]nipecotic acid showed that the radioactivity was associated mostly with the satellite glial cells, but silver grains were also found over the sensory fibres and to a small extent over the neurones (Fig. 3a,b). Control ganglia processed for autoradiography and exposed for the same time as the radioactive sections showed no positive chemography ; however control sections on which the emulsion had been fogged showed occasional patches where the silver grain density was low. This may have been due to negative chemography, despite the celloidin layer, or to thinning of the emulsion over humps in the sections; the patches occurred indiscriminately over the tissue, which would argue against selective negative chemography. This pattern was only seen infrequently over the radioactive sections, and therefore the distribution of grains seen in Fig. 3 is considered to represent the true distribution of carbon-14 in the tissue. The retention of [14C]nipecotic acid by the ganglia following fixation with either glutaraldehyde or acrolein was very poor, being only 6”/, and 12%, respectively. These levels are, of course, unacceptably low for autoradiographic purposes since there is a possibility that retained material may be preferentially localised in a way unrelated to the original distribution of radioactivity in the tissue. Nevertheless, autoradiographs were obtained following acrolein fixation after a very long exposure, and are presented here since they compare favourably with those obtained from frozen ganglia (Fig. 3c,d). Again, silver grains were seen principally over satellite glia, while the sensory fibres and neurones appeared to be labelled to a similar extent. Tests for positive and negative chemography showed that neither had occurred. Some autoradiographs were obtained from ganglia incubated at 25°C and thcse showed a similar distribution of radioactivity to that found at 37°C. DISCUSSION
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potency of the other inhibitors of the uptake of nipecotic acid into sensory ganglia is similar to that for nipecotic acid uptake into brain slices, and none of the compounds which failed to inhibit nipecotic acid uptake into the ganglia influenced its uptake into et al., 1976~). brain slices, and vice versa (JOHNSTON One exception to this was p-mercuriphenylsulphonate which was less effective as an inhibitor of nipecotic acid uptake into sensory ganglia than into brain slices, as was found for GABA uptake into these two tissues (IVERSEN & JOHNSTON, 1971; JOHNSTON et al., 1976a; SCHON& KELLY,1974b). It appears therefore that nipecotic acid is transported by the same glial system that mediates GABA uptake into sensory ganglia, and the light microscope autoradiographs presented here would lend some support to this. However, the labelling of fibre tracts and neurones was greater following [‘‘C]nipecotic acid uptake than after C3H]GABA uptake into the ganglia (SCHON& KELLY,1974~).This may be due to the lower affinity of the nipecotic acid uptake process, since the difference between the localisation of the high affinity and the less specific, low affinity uptake process would become less marked. As SCHON & KELLY(1974b) have shown, the distribution of radioactivity following low affinity uptake of C3H]GABA into sensory ganglia is similar to that found in this study for nipecotic acid. It is interesting to note that while nipecotic acid is transported by the GABA system in both neurones and glia it has less affinity than GABA for the glial system and a greater affinity than GABA for the neuronal system. Moreover (-)-nipecotic acid, which has an affinity for the neuronal GABA carrier approx 5 times higher than the (+) isomer, has a configuration remarkably similar to that of (+)-DABA and like the latter shows a marked selectivity towards the neuronal GABA uptake system (JOHNSTONet ul., 1976b). BOWERYet a[. (1976) have similarly shown that (-k) nipecotic acid is more effective as an inhibitor of GABA uptake into cortical slices (‘neuronal uptake’) than into superior cervical ganglia (‘glial uptake’). It may be inferred therefore that the folded conformation and interionic distance of nipecotic acid (JOHNSTON et ul., 1 9 7 6 ~ are ) complementary to the carrier site in nerve endings, while the carrier site on glial cells has slightly different requirements. Since p-alanine is an inhibitor of both GABA and nipecotic acid uptake into glia and has a charge separation which is similar to that of nipecotic acid (JOHNSTON et al., 1976u), it appears that the charge separation of nipecotic acid is satisfactory but that its conformation does not match so well the configuration of the glial GABA carrier site.
The uptake of [‘4C]nipecotic acid displays similar saturable kinetics to the uptake of C3H]GABA into dorsal root ganglia, although it does seem to have a lower affinity constant (SCHON& KELLY, 1974b). This may in part explain the lower tissue:medium ratios achieved by nipecotic acid, although the comparative figures for GABA were obtained at a higher temperature (SCHON& KELLY,1974b). In addition, the similarity between nipecotic acid and GABA uptake is shown by the fact that in sensory ganglia fl-alanine is considerably more effective as an inhibitor of both uptake systems than is REFERENCES L-DABA. In small brain slices L-DABA is a more powerful inhibitor of the uptake of both nipecotic BOWERYN. G., JONES G. P.& NEALM. J. (1976) Selective acid and GABA than is P-alanine (IVERSEN& JOHNinhibition of neuronal GABA uptake by cis-1,3-aminocyclohexane carboxylic acid. Nature, Lond. 264, 281-284. STON, 1971; JOHNSTONet al., 1976~).The order of
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M .c. w. MINCHIN
IVERSEN L. L. & JOHNSTON G. A. R. (1971) GABA uptake acid, various isoxazoles and related compounds. J . in rat central nervous system: comparison of uptake in Neurochem. 25, 797-802. slices and homogenates and the effects of some inhibi- KROGSGAARD-LARSEN P., JOHNSTON G. A. R., CURTISD. R., tors. J. Neurochern. 18, 1939-1950. GAMEC. J. A. & MCCULLOCH R . M. (1975) Structure IVERSENL. L. & KELLYJ. S. (1975) Uptake and metabolism and biological activity of a series of conformationally of y-aminobutyric acid by neurones and glial cells. Biorestricted analogues of GABA. J . Neurochem. 25, 80-V chem. Pharmac. 24, 933-938. 809. JOHNSTON G . A. R.,STEPHANSON A. I>. & TWITCHIN B. MINCHINM. C. W. & IVERSEN L. L. (1974) Release of (19760) Uptake and release of nipecotic acid by rat brain [3H]gamma-aminobutyric acid from glial cells in rat slices. J. Neurochem. 26, 83-87. dorsal root ganglia. J . Neurochem. 23, 533-540. JOHNSTON G . A. R., KROGSGAARD-LARSEN P., STEPHANSONSCHON F. & KELLY J. S. (19740) Autoradiographic localisaA. L. & TWITCHIN B. (19766) Inhibition of the uptake tion of 13H]GABA and [3H]glutamate over satellite glial cells. Brain Res. 66, 275-288. of GABA and related amino acids in rat brain slices by the optical isomers of nipecotic acid. J . Neurochem. SCHONF. & KELLYJ. S. (1974b) The characterisation of 26, 1029-1032. ['HIGABA uptake into the satellite glial cells of rat KROGSGAARD-LARSEN P. &JOHNSTON G. A. R. (1975) Inhisensory ganglia. Brain Res. 66, 289-300. bition of GABA uptake in rat brain slices by nipecotic
