0022-3042/79/020 1-0669502 0010
Joirrnrrl of Nrarocharr~~siryVol 32. pp. 669-672 Pergamon Press Ltd 1979. Printed in Great Britain Q lnterndtional Society for Neurochemlstry Lrd
SHORT COMMUNICATION
The role of pH in the isolation of nerve ending particles which transport GABA and glutamic acid (Received 12 M a y 1978 Accepted 23 June 1978)
RECENTLY we have examined various structural, topographical and developmental aspects of CABA transport into rat brain nerve ending particles (HITZEMANN& LOH, 1978a, b, c), During the course of these studies, we observed that the yield of viable nerve endings, as measured by the rate of labeled GABA transport, varied significantly from preparation to preparation. In an attempt to locate the cause or causes of these variations, we began a systematic investigation of the technique used to prepare the nerve endings. Our initial results indicated that the p H of the sucrose solutions used in the various isolation procedures was one of the single most critical factors in determining the viable nerve ending yield. We, therefore, undertook a detailed investigation of the role p H plays in preparing nerve endings that are used to measure labeled GABA transport. Our results, as described in this report, suggest that a p H of 7.8 is optimum for preparing such nerve endings. MATERIALS AND METHODS Materials. [2-'H]GABA (spec. act. 43 Ci/mmol). [2-3H]fl-alanine (spec. act. 39 Ci/mmol), [U-'4C]glutamic acid (spec. act. 200 mCi/mmol) and [3H-G]ouabain (spec. act. 14.4 Ci/mmol) were obtained from New England Nuclear Corporation, Boston, MA. All special biochemicals were purchased from Sigma Chemical Co., St. Louis, MO. Nerve ending preparation. Sprague -Dawley rats (Simonsen Laboratories, Gilroy, CA) weighing 200-300 g were used in all experiments. The animals were killed by decapitation. The brains were removed, weighed and then homogenized in 19 vol of 0.32 M-sucrose + 5 mM-HEPES (S-H). The S-H solution was adjusted with NaOH to the desired pH. Thirty ml of the homogenate was centrifuged for 10inin at lOOOg t o remove a crude nuclear fraction. The supernatant was transferred and centrifuged for 10 min at 17,000 g. The crude mitochondrial-nerve ending pellet was then washed twice using 30ml of S-H. The washed pellet was next resuspended in 7 ml of S-H and layered on a four step discontinuous Ficoll-S-H gradient consisting of 7 ml each 15, 12, 8 and 6%, Ficoll in S-H. The gradient was centrifuged for 45 min at 25,00Orev./min in a Spinco SW-27 rotor. After centrifugation, 5 fractions were removed from the gradient. Each fraction (A-E) conAhbreurattons used: S-H, 0.32 M-sucrosc + 5 mMHEPES; KRB, Krebq-Ringer-bicarbonate; HEPES, N-2hydroxyethylpiperazine-N'-2-ethane-sulfonicacid.
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sisted of the material at the interface (or the pellet) and the material suspended in solution above the interface (see Fig. 1). The fractions were diluted to 40ml with S-H and centrifuged for IOmin at 45,000g. The pellets obtained were resuspended in 2 m l of S-H, pH = 7.4 and used immediately for the measurement of GABA and glutamate transport. The total time from the initial homogenization to the measurement of transport was between 3 and 4 h. In order to ensure that there was n o delay in processing the tissue, the gradients were prepared prior to killing the animals. G A B A and glutarnate transport. One hundred microliters of fractions A and E, 50 pI of fraction B or 25 p1 of fractions C and D were added to 6 m l polypropylene miniscintillation vials (Fischer Chemicals Co.) which contained 0.8 ml Krebs-Ringer-bicarbonate (KRB) buffer and sufficient S-H to make the final volume 0.9ml. The KRB buffer contained, in addition to the normal salts, IOmMHEPES and 10 mwglucose. This mixture was preincubated at 27°C for 5 min prior to the addition of 2 x M-[~H]GABAand 8 x lo-" ~ - [ ' ~ C ] g l u t a m a t e(final concentrations). These concentrations of [3H]GABA and ['4C]glutamate werc previously determined to be the K, (or K , ) concentrations (HITZEMANN & LDH, in press). The incubation was continued for 5min before cooling the samples for 5min in an ice-bath. The samples were then centrifuged for 10 min at 45,000 g. The supernatant was aspirated away and the surface of the pellet was gently washed with approx 2 m l of buffer. The pellet was then vigorously suspended in 0.5 in1 of 0.1% SDS prior to the addition of 4.5 ml of Scintiverse cocktail (Fischer Chemical Co.). The samples were counted 24 h after adding the cocktail in a Beckman LS-100 scintillation counter. Blanks, prepared by replacing the sodium in the KRB buffer with isotonic amounts of sucrose, demonstrated that more than 99% of the transport was sodium dependent. Under the conditions stated, transport increased linearly for the 5 min incubation period and the amounts of tissue used were within the linear range. Ouahnin binding. An aliquot of each fraction was frpzen at -20°C and saved (48-72 h) for the measurement of ouabain binding. The freezing and thawing procedure resulted in less than a 5% loss of binding activity. Ouabain binding was measured using a modified procedure based on the methods described by HARRISe f al. (1973) and GELBART & GOLDMAN (1977): The reaction buffer contained (final concentrations) 100 mM-NaC1, 3 mM-MgCl,, 3 mM-ATP and SO mM-HEPES buffcred to p H = 7.4 at 27°C with Tris. This mixture was preincubated for 5 min at 27°C in a 6 ml mini-scintillation vial prior to the addition of 100 p1 of
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the fraction to be examined. The incubation was continued for 2 min before adding ['Hlouabain (final concentration, M). The final reaction volume was 1 ml. Exactly 2 x 2 min later, the reaction was stopped by rapid cooling in an ice bath. After 5 m i n the samples were centrifuged, washed and counted as described for the transport experiments. Blanks were prepared for all samples by measuring binding in the absence of NaCI, MgC1, and ATP. Under the conditions described, binding increased linearly during the 2 min incubation period and the amounts of tissue were within the linear range. NADPH-cytochrorne c reductase and succinic dehydrogenuw activity. N A D P H cytochrome c reductase activity was assayed as described by SOTTOCASA et a\. (1974). Succinic dehydrogenase was measured as described by EARL & KORMER(1965). Protein was measured as described by LOWRY et al. (1951) using bovine serum albumin as the standard. Statistical analysis was performed using Student's t-test.
A
RESULTS Effect of p H on the total activity and specific activity of ['HIGABA and ['4C]glutamate transport sites within the various fractions of the Ficoll-S-H gradient In a series of preliminary experiments, brains were homogenized in S-H at pH values of 6.0, 6.4, 6.8 and 7.0 and processed at the same p H throughout all procedures including the density gradient centrifugation. At all p H values below 7, the amount of ['HIGABA transport activity recovered in the various fractions was markedly decreased. For example, at p H = 6.8, the total transport ac-
Froction
FIG. 1. Effect of pH on ['HIGABA transport into various fractions of the Ficoll-S-H gradient. Fractions A-E (see inset) were prepared using S-H solutions varying from p H = 7.0 t o p H = 8.2. After harvesting, the fractions were M, final concenthen analyzed for ['HIGABA (2 x tration) transport activity using a centrifugation assay. The data are presented in terms of total activity per fraction (nmoles C3H]GABA transported/5 min) and in terms of specific activity (nmoles ['HIGABA transported/5 min/mg protein). All data are the mean & S.E. of 3-4 experiments. *-Significantly different from p H = 7 value, P < 0.05.
A
0
C
D
E
Fraction
I%.
2. Effect of p H on [14C]glutamate transport into various fractions of the Ficoll-S-H gradient. The experimental details are the same as in the legend to Fig. 1 except that [14C]glutamate (final concentration 8 x 1 0 - 6 ~ ) transport was measured. The concentration (8 x 1 0 - 6 ~ ) of ['4C]glutamate used was previously dctcrniined to bc & LOH, 1978~).Data the K , concentration (HJTZEMANN are thc mean k S.E. of three experiments. *--Significantly different from pH = 7 value, P < 0.05. tivity recovered from the gradient was 56% of the activity recovered when at a pH of 7. Since from these data it was obvious that the use of S-H solutions below a p H of 7 was inappropriate for preparing viable nerve endings, our attention turned to the use of S-H solutions at a p H of 7 and above. A detailed investigation of the isolation procedure was made at p H = 7, 7.4, 7.8 and 8.2. The data in Fig. 1A show that as compared t o the levels at p H = 7, significant increases in total C3H]GABA transport activity were obtained in fraction C at p H = 7.4, 7.8 and 8.2 and in fraction D at p H = 7.8 and in fractions A and B at p H = 8.2. In general, these increases were also reflected by increases in specific activity (Fig. IB). However, the changes in specific activity in fraction C were not as large as those in total activity since the amount of protein appearing in this fraction increased with increasing pH. At p H = 8.2, the increase in fraction C protein was large enough (4.52 f 0.18 mg proteinbrain vs 2.83 k 0.30 mg proteinlbrain) so that there was n o significant increase in fraction C specific activity. In some experiments [14C]glutamate transport was measured simultaneously with C3H)GABA transport and the results of these'studies are shown in Fig. 2. The pattern of p H induced changes in ['4C]glutamate transport was somewhat different than the changes observed for C3H]GABA transport. For example, both the total transport and transport specific activity of fraction A was increased over the p H = 7 value at p H = 7.4, 7.8 and 8.2. Also unlike the ['HJGABA data, the total and specific activity of fraction B were significantly increased at p H = 7.8 over the p H = 7 values. Other changes that occurred in [14C]glutamate transport as a function of pH were similar t o those seen for C3H]GABA transport. Over-
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the amount of glial vesicles in fraction C was tested indirectly by measuring high afinity 13H]P-alanine transport (see HITZEMANN & LOH, 1978a). At ail pH values the specific activity of [3H]fl-alanine transport sites was between 7 and lo%, of the values for C3H]GABA transport. Although it was previously concluded that [3H]fi-alanine transport occurs primarily in nerve endings, the measurement of ['HID-alanine transport can serve to indicate the maximum level of glial vesicle contamination. Previous work by SCHON& KCLLEY (1974) has demonstrated the high afinity of B-alanine for glial GABA transport sites.
. DISCUSSION
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Although the physiological significance of the high affinity GABA transport system in the CNS remains unclear (IVERSEN,1975; LEVI & RAITERI,1975; ROSKOSKI, a 1977), its usefulness as a tool to label and localize nerve a B C D E endings in tissue homogenates remains unquestioned. PreFraction & vious studies have demonstrated directly (TVERSEN FIG.3. Effect of p H on ['Hlouabain binding activity to BLOOM,1972) and indirectly (MARTIN,1976; HOKFELDTet 1969; the various fractions of the Ficoll S-€I gradient. The ex- al., 1970; WOFSEYer al., 1971; NEAI. & IVERSEN, perimental details are the same as in thc legend to Fig. SNYDERe / al., 1970; BENNETTet al., 1973) that the high 1, except that [3H]ouabain (final concentration, 2 x 1 0 - 8 ~ ) affinity transport of labeled GABA (or glutamic acid) in binding was measured using the centrifugation assay de- tissue slices or homogenates occurs primarily in nerve scribed in the Methods. Data are expressed both as total endings. However. in order to effectively capitalize on binding per fraction (pmol bound/5 min) and as specific using the high affinity transport of GABA to label nerve activity (pmol bound/5 min) and as spccific activity (pmol endings, it is necessary to know the optimum experimental bound/5 minimg protein). Data are the mean S.E. of conditions for labeling. The results of the present study reveal that the optimum thrcc experimcnts, each performed in duplicate. *-Signifip H for preparing nerve endings to be used in 13H]GABA cantly different from control, P < 0.05. and ['4C]glutamate transport studies is about p H = 7.8. The conditions were.not maximal at p H = 7.4 and thus all, the data shown in Figs. 1 and 2 illustrate that increas- it cannot be assumed that the use of solutions buffered ing the pH of the S-H solution from 7 to 7.8 provides to physiological p H will provide the ideal p H for preparing viable nerve endings. These effects of changing the p H were a significant advantage for the measurement of ['HIGABA particularly obvious in fraction C, the fraction most comand ['4C]glutamate transport. monly harvested for 'pure' nerve endings. The changes in Effect of p H on [3H]ouabaiii binding within various fractions total activity in this fraction were greater than those in of the Ficoll-S-H gradient specific activity which suggests that as the p H was inThe data in Fig. 3 illustrate that ['Hlouabain binding creased non-nerve ending organelles infiltrated the fraction. sites, like the ['HIGABA and ['4C]glutamate transport This conclusion was borne out by the measurement of sucsites, were significantly influenced by changes in S-H pH. cinic dehydrogenase and N A D P H cytochrome c reductase In particular, both the total and specific activity of the activity in fraction C. It was found that the total activity binding sites in fractions A and B were significantly in- of these enzymes increased with increasing pH. However, creased as the p H was increased from 7 to 7.8. However, the specific activity of the enzymes did not significantly in fracti-on C increasing the p H did not increase the specific increase, since the increases in total enzyme activity were activity of the C3H]ouabain binding sites. Although the comparable in magnitude to the increases in protein contotal binding activity was increased in this fraction as the tent of fraction C. This is different from the effects on transp H increased, the magnitude of these changes was not port, where both total and specific activity increased. Thus, increasing the p H of the S-H solutions to 7.8 exerted a greater than the increases in protein levels. selective effect on maintaining and/or increasing transport Eflect o f p H on rhe conrainination offraction C viability. Further evidence of this selectivity was seen in The data in Figs. 1 and 2 suggest that increasing the the C3H]ouabain binding data. Although increasing the pH p H of the S-H solution results in the recovery of more above 7 increased the specific activity of binding sites in viable nerve endings in fraction C. It was of interest to fractions A and B, n o significant change was observed in know if this increase in viability was associated with fraction C . Thus, it cannot be assumed that all membranc changes in the mitochondrial and membranous contami- functions or activities will be equally affected (or protected) by changes in the p H of the sucrose solutions. nation. It was found that increasing the pH of the S-H solution has n o effect on the specific activity of succinic dehydrogenase or NADPH-cytochrome c reductase in fraction C which suggests that the levels of mitochondrial and microsonial contamination are not affected by changes Acknowledgements-The author wishes to acknowledge the in pH. The possibility that increasing the p H increased technical assistance of ELAINERYAN and the editorial and
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Regional uptake and subcellular localization of [3H]gamma-aminobutyric acid (GABA) in brain slices. Life Sci. (I) 9, 203-212. Depnr[tiien!s of' Psychiatry and ROBERTJ. HITZEMANNIVERSEN L. L. & BLOOMF. E. (1972) Studies on the uptake Pharmacology, of ['HIGABA and ['Hlglycine by slices and homogenates of rat brain and 'spinal cord by electron microUniversity of California, scopic autoradiography. Brain Rex 41, 131-143. San Francisco, C A 94143, IVERSEN L. (1975) High affinity uptake of neurotransmitter USA. amino acids. Narure 253, 481. LEV] G., BERLOTTINI A,, CHENJ. & RAITERI M. (1974) REFERENCES Regional differences in the synaptosomal uptake of 'H-y-aminobutyric acid and '*C-glutamate and possible BENNETT J. P., JR., LOGANW. J. & SNYDER S. H. (1973) role of exchange processes. J . Pharmac. exp. Ther. 188, Amino acids as central nervous transmitters. The in429-438. flucnce of ions, amino acid analogues, and ontogeny on transport systems for L-glutamic and L-aspartic acids LEVIG. & RAITERIM. (1975) Reply. Nature 253, 481-482. 0. H., ROSERROUGHN. J., FARR A. L. & RANDALL and glycine into central nervous synaptosomes of the LOWRY R. J. (1951) Protein measurement with the Fohn phenol rat. J . Neurochem 21, 1533-1550. reagent. J . biol. Chem. 193, 265-275. A. (1965) The isolation and EARLD. C. N. & KORMER L. L. (1969) Subcellular distribution properties of cardiac ribosomes and polysomes. Biochern. NEALM. J. & IVERSEN of endogenous and L3H]-y-aminobutyric acid from brain J . 94, 721-734. slices. J. Neuroc!ient. 16, 1245-1252. GELRART A. & GOLDMAN R. (1977) Correlation between ROSKOSKI R. (1977) Net uptake of L-glutamate and GABA microsomal Na*, K + ATPase activity and L'H]ouabain by high affinity synaptosomal transport systems. Soc. binding to heart tissue homogenates. Biochitn. biophys. Neurosci. Abstr. 7, 1315. Acta 481, 689494. J. S. (1974) Autoradiographic localizaHARRIS W. E., SWANSON P. D. & STAHLW. L. (1973) Oua- SCHONF. & KELLEY tion of 13H]GABA and L3H]glutamate over satellite bain binding sites and the (Na+, K+)-ATPase of brain glial cells. Brain Rex 66, 275-278. microsomal membranes. Biochini. biophys. Acta 298, SNYDER S. H., KUHARM. J., GREENA. I., COYLEJ. T. 68M89. & SHASKAN E. G. (1970) Uptake and subcellular localiHITZEMANN R. J. & LOH H. H. (1978~)A comparison of zation of neurotransmitters in the brain. Inr. Rev. NeuroGABA and 8-alanine transport and GABA membrane bid. 13, 127-158. binding in the rat brain. J. Neurochem. 30, 471477. G . L., KIJYLENSTEIRNA B., ERNSTER L. & BERG R. J. & LOH H. H. (197%) Effects of some SOTTOCASA HITZEMANN STAND A. (1974) An electron transport system associated conformationally restricted GABA analogues on GABA membrane binding and nerve ending transport. Brain with the outer membrane of liver mitochondria. J . Cell. B i d . 32, 41 5-438. R ~ s 144, . 163-169. M. J. & SNVDER S. H. (1971) A HITZEMANN R. J. & LOH H. H. High affinity GABA WOFSEYA. R., KCJHAR unique synaptosomal fraction which accumulates glutaand glutamate transport in developing nerve ending parmic and aspartic acids in brain tissue. Proc. narn. Acad. ticles. Brain Res., in press. Sci., U.S.A. 68, 1102-1106. A. (1970) HOKFELDTT., JONSON G. & LYUNGUAHL typing assistance of KAYEWELCH.This study was supported in part by N.I.N.C.D.S. Grant NS-13398.
Joirrnrrl of Nrarocharr~~siryVol 32. pp. 669-672 Pergamon Press Ltd 1979. Printed in Great Britain Q lnterndtional Society for Neurochemlstry Lrd
SHORT COMMUNICATION
The role of pH in the isolation of nerve ending particles which transport GABA and glutamic acid (Received 12 M a y 1978 Accepted 23 June 1978)
RECENTLY we have examined various structural, topographical and developmental aspects of CABA transport into rat brain nerve ending particles (HITZEMANN& LOH, 1978a, b, c), During the course of these studies, we observed that the yield of viable nerve endings, as measured by the rate of labeled GABA transport, varied significantly from preparation to preparation. In an attempt to locate the cause or causes of these variations, we began a systematic investigation of the technique used to prepare the nerve endings. Our initial results indicated that the p H of the sucrose solutions used in the various isolation procedures was one of the single most critical factors in determining the viable nerve ending yield. We, therefore, undertook a detailed investigation of the role p H plays in preparing nerve endings that are used to measure labeled GABA transport. Our results, as described in this report, suggest that a p H of 7.8 is optimum for preparing such nerve endings. MATERIALS AND METHODS Materials. [2-'H]GABA (spec. act. 43 Ci/mmol). [2-3H]fl-alanine (spec. act. 39 Ci/mmol), [U-'4C]glutamic acid (spec. act. 200 mCi/mmol) and [3H-G]ouabain (spec. act. 14.4 Ci/mmol) were obtained from New England Nuclear Corporation, Boston, MA. All special biochemicals were purchased from Sigma Chemical Co., St. Louis, MO. Nerve ending preparation. Sprague -Dawley rats (Simonsen Laboratories, Gilroy, CA) weighing 200-300 g were used in all experiments. The animals were killed by decapitation. The brains were removed, weighed and then homogenized in 19 vol of 0.32 M-sucrose + 5 mM-HEPES (S-H). The S-H solution was adjusted with NaOH to the desired pH. Thirty ml of the homogenate was centrifuged for 10inin at lOOOg t o remove a crude nuclear fraction. The supernatant was transferred and centrifuged for 10 min at 17,000 g. The crude mitochondrial-nerve ending pellet was then washed twice using 30ml of S-H. The washed pellet was next resuspended in 7 ml of S-H and layered on a four step discontinuous Ficoll-S-H gradient consisting of 7 ml each 15, 12, 8 and 6%, Ficoll in S-H. The gradient was centrifuged for 45 min at 25,00Orev./min in a Spinco SW-27 rotor. After centrifugation, 5 fractions were removed from the gradient. Each fraction (A-E) conAhbreurattons used: S-H, 0.32 M-sucrosc + 5 mMHEPES; KRB, Krebq-Ringer-bicarbonate; HEPES, N-2hydroxyethylpiperazine-N'-2-ethane-sulfonicacid.
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sisted of the material at the interface (or the pellet) and the material suspended in solution above the interface (see Fig. 1). The fractions were diluted to 40ml with S-H and centrifuged for IOmin at 45,000g. The pellets obtained were resuspended in 2 m l of S-H, pH = 7.4 and used immediately for the measurement of GABA and glutamate transport. The total time from the initial homogenization to the measurement of transport was between 3 and 4 h. In order to ensure that there was n o delay in processing the tissue, the gradients were prepared prior to killing the animals. G A B A and glutarnate transport. One hundred microliters of fractions A and E, 50 pI of fraction B or 25 p1 of fractions C and D were added to 6 m l polypropylene miniscintillation vials (Fischer Chemicals Co.) which contained 0.8 ml Krebs-Ringer-bicarbonate (KRB) buffer and sufficient S-H to make the final volume 0.9ml. The KRB buffer contained, in addition to the normal salts, IOmMHEPES and 10 mwglucose. This mixture was preincubated at 27°C for 5 min prior to the addition of 2 x M-[~H]GABAand 8 x lo-" ~ - [ ' ~ C ] g l u t a m a t e(final concentrations). These concentrations of [3H]GABA and ['4C]glutamate werc previously determined to be the K, (or K , ) concentrations (HITZEMANN & LDH, in press). The incubation was continued for 5min before cooling the samples for 5min in an ice-bath. The samples were then centrifuged for 10 min at 45,000 g. The supernatant was aspirated away and the surface of the pellet was gently washed with approx 2 m l of buffer. The pellet was then vigorously suspended in 0.5 in1 of 0.1% SDS prior to the addition of 4.5 ml of Scintiverse cocktail (Fischer Chemical Co.). The samples were counted 24 h after adding the cocktail in a Beckman LS-100 scintillation counter. Blanks, prepared by replacing the sodium in the KRB buffer with isotonic amounts of sucrose, demonstrated that more than 99% of the transport was sodium dependent. Under the conditions stated, transport increased linearly for the 5 min incubation period and the amounts of tissue used were within the linear range. Ouahnin binding. An aliquot of each fraction was frpzen at -20°C and saved (48-72 h) for the measurement of ouabain binding. The freezing and thawing procedure resulted in less than a 5% loss of binding activity. Ouabain binding was measured using a modified procedure based on the methods described by HARRISe f al. (1973) and GELBART & GOLDMAN (1977): The reaction buffer contained (final concentrations) 100 mM-NaC1, 3 mM-MgCl,, 3 mM-ATP and SO mM-HEPES buffcred to p H = 7.4 at 27°C with Tris. This mixture was preincubated for 5 min at 27°C in a 6 ml mini-scintillation vial prior to the addition of 100 p1 of
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the fraction to be examined. The incubation was continued for 2 min before adding ['Hlouabain (final concentration, M). The final reaction volume was 1 ml. Exactly 2 x 2 min later, the reaction was stopped by rapid cooling in an ice bath. After 5 m i n the samples were centrifuged, washed and counted as described for the transport experiments. Blanks were prepared for all samples by measuring binding in the absence of NaCI, MgC1, and ATP. Under the conditions described, binding increased linearly during the 2 min incubation period and the amounts of tissue were within the linear range. NADPH-cytochrorne c reductase and succinic dehydrogenuw activity. N A D P H cytochrome c reductase activity was assayed as described by SOTTOCASA et a\. (1974). Succinic dehydrogenase was measured as described by EARL & KORMER(1965). Protein was measured as described by LOWRY et al. (1951) using bovine serum albumin as the standard. Statistical analysis was performed using Student's t-test.
A
RESULTS Effect of p H on the total activity and specific activity of ['HIGABA and ['4C]glutamate transport sites within the various fractions of the Ficoll-S-H gradient In a series of preliminary experiments, brains were homogenized in S-H at pH values of 6.0, 6.4, 6.8 and 7.0 and processed at the same p H throughout all procedures including the density gradient centrifugation. At all p H values below 7, the amount of ['HIGABA transport activity recovered in the various fractions was markedly decreased. For example, at p H = 6.8, the total transport ac-
Froction
FIG. 1. Effect of pH on ['HIGABA transport into various fractions of the Ficoll-S-H gradient. Fractions A-E (see inset) were prepared using S-H solutions varying from p H = 7.0 t o p H = 8.2. After harvesting, the fractions were M, final concenthen analyzed for ['HIGABA (2 x tration) transport activity using a centrifugation assay. The data are presented in terms of total activity per fraction (nmoles C3H]GABA transported/5 min) and in terms of specific activity (nmoles ['HIGABA transported/5 min/mg protein). All data are the mean & S.E. of 3-4 experiments. *-Significantly different from p H = 7 value, P < 0.05.
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C
D
E
Fraction
I%.
2. Effect of p H on [14C]glutamate transport into various fractions of the Ficoll-S-H gradient. The experimental details are the same as in the legend to Fig. 1 except that [14C]glutamate (final concentration 8 x 1 0 - 6 ~ ) transport was measured. The concentration (8 x 1 0 - 6 ~ ) of ['4C]glutamate used was previously dctcrniined to bc & LOH, 1978~).Data the K , concentration (HJTZEMANN are thc mean k S.E. of three experiments. *--Significantly different from pH = 7 value, P < 0.05. tivity recovered from the gradient was 56% of the activity recovered when at a pH of 7. Since from these data it was obvious that the use of S-H solutions below a p H of 7 was inappropriate for preparing viable nerve endings, our attention turned to the use of S-H solutions at a p H of 7 and above. A detailed investigation of the isolation procedure was made at p H = 7, 7.4, 7.8 and 8.2. The data in Fig. 1A show that as compared t o the levels at p H = 7, significant increases in total C3H]GABA transport activity were obtained in fraction C at p H = 7.4, 7.8 and 8.2 and in fraction D at p H = 7.8 and in fractions A and B at p H = 8.2. In general, these increases were also reflected by increases in specific activity (Fig. IB). However, the changes in specific activity in fraction C were not as large as those in total activity since the amount of protein appearing in this fraction increased with increasing pH. At p H = 8.2, the increase in fraction C protein was large enough (4.52 f 0.18 mg proteinbrain vs 2.83 k 0.30 mg proteinlbrain) so that there was n o significant increase in fraction C specific activity. In some experiments [14C]glutamate transport was measured simultaneously with C3H)GABA transport and the results of these'studies are shown in Fig. 2. The pattern of p H induced changes in ['4C]glutamate transport was somewhat different than the changes observed for C3H]GABA transport. For example, both the total transport and transport specific activity of fraction A was increased over the p H = 7 value at p H = 7.4, 7.8 and 8.2. Also unlike the ['HJGABA data, the total and specific activity of fraction B were significantly increased at p H = 7.8 over the p H = 7 values. Other changes that occurred in [14C]glutamate transport as a function of pH were similar t o those seen for C3H]GABA transport. Over-
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the amount of glial vesicles in fraction C was tested indirectly by measuring high afinity 13H]P-alanine transport (see HITZEMANN & LOH, 1978a). At ail pH values the specific activity of [3H]fl-alanine transport sites was between 7 and lo%, of the values for C3H]GABA transport. Although it was previously concluded that [3H]fi-alanine transport occurs primarily in nerve endings, the measurement of ['HID-alanine transport can serve to indicate the maximum level of glial vesicle contamination. Previous work by SCHON& KCLLEY (1974) has demonstrated the high afinity of B-alanine for glial GABA transport sites.
. DISCUSSION
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Although the physiological significance of the high affinity GABA transport system in the CNS remains unclear (IVERSEN,1975; LEVI & RAITERI,1975; ROSKOSKI, a 1977), its usefulness as a tool to label and localize nerve a B C D E endings in tissue homogenates remains unquestioned. PreFraction & vious studies have demonstrated directly (TVERSEN FIG.3. Effect of p H on ['Hlouabain binding activity to BLOOM,1972) and indirectly (MARTIN,1976; HOKFELDTet 1969; the various fractions of the Ficoll S-€I gradient. The ex- al., 1970; WOFSEYer al., 1971; NEAI. & IVERSEN, perimental details are the same as in thc legend to Fig. SNYDERe / al., 1970; BENNETTet al., 1973) that the high 1, except that [3H]ouabain (final concentration, 2 x 1 0 - 8 ~ ) affinity transport of labeled GABA (or glutamic acid) in binding was measured using the centrifugation assay de- tissue slices or homogenates occurs primarily in nerve scribed in the Methods. Data are expressed both as total endings. However. in order to effectively capitalize on binding per fraction (pmol bound/5 min) and as specific using the high affinity transport of GABA to label nerve activity (pmol bound/5 min) and as spccific activity (pmol endings, it is necessary to know the optimum experimental bound/5 minimg protein). Data are the mean S.E. of conditions for labeling. The results of the present study reveal that the optimum thrcc experimcnts, each performed in duplicate. *-Signifip H for preparing nerve endings to be used in 13H]GABA cantly different from control, P < 0.05. and ['4C]glutamate transport studies is about p H = 7.8. The conditions were.not maximal at p H = 7.4 and thus all, the data shown in Figs. 1 and 2 illustrate that increas- it cannot be assumed that the use of solutions buffered ing the pH of the S-H solution from 7 to 7.8 provides to physiological p H will provide the ideal p H for preparing viable nerve endings. These effects of changing the p H were a significant advantage for the measurement of ['HIGABA particularly obvious in fraction C, the fraction most comand ['4C]glutamate transport. monly harvested for 'pure' nerve endings. The changes in Effect of p H on [3H]ouabaiii binding within various fractions total activity in this fraction were greater than those in of the Ficoll-S-H gradient specific activity which suggests that as the p H was inThe data in Fig. 3 illustrate that ['Hlouabain binding creased non-nerve ending organelles infiltrated the fraction. sites, like the ['HIGABA and ['4C]glutamate transport This conclusion was borne out by the measurement of sucsites, were significantly influenced by changes in S-H pH. cinic dehydrogenase and N A D P H cytochrome c reductase In particular, both the total and specific activity of the activity in fraction C. It was found that the total activity binding sites in fractions A and B were significantly in- of these enzymes increased with increasing pH. However, creased as the p H was increased from 7 to 7.8. However, the specific activity of the enzymes did not significantly in fracti-on C increasing the p H did not increase the specific increase, since the increases in total enzyme activity were activity of the C3H]ouabain binding sites. Although the comparable in magnitude to the increases in protein contotal binding activity was increased in this fraction as the tent of fraction C. This is different from the effects on transp H increased, the magnitude of these changes was not port, where both total and specific activity increased. Thus, increasing the p H of the S-H solutions to 7.8 exerted a greater than the increases in protein levels. selective effect on maintaining and/or increasing transport Eflect o f p H on rhe conrainination offraction C viability. Further evidence of this selectivity was seen in The data in Figs. 1 and 2 suggest that increasing the the C3H]ouabain binding data. Although increasing the pH p H of the S-H solution results in the recovery of more above 7 increased the specific activity of binding sites in viable nerve endings in fraction C. It was of interest to fractions A and B, n o significant change was observed in know if this increase in viability was associated with fraction C . Thus, it cannot be assumed that all membranc changes in the mitochondrial and membranous contami- functions or activities will be equally affected (or protected) by changes in the p H of the sucrose solutions. nation. It was found that increasing the pH of the S-H solution has n o effect on the specific activity of succinic dehydrogenase or NADPH-cytochrome c reductase in fraction C which suggests that the levels of mitochondrial and microsonial contamination are not affected by changes Acknowledgements-The author wishes to acknowledge the in pH. The possibility that increasing the p H increased technical assistance of ELAINERYAN and the editorial and
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Regional uptake and subcellular localization of [3H]gamma-aminobutyric acid (GABA) in brain slices. Life Sci. (I) 9, 203-212. Depnr[tiien!s of' Psychiatry and ROBERTJ. HITZEMANNIVERSEN L. L. & BLOOMF. E. (1972) Studies on the uptake Pharmacology, of ['HIGABA and ['Hlglycine by slices and homogenates of rat brain and 'spinal cord by electron microUniversity of California, scopic autoradiography. Brain Rex 41, 131-143. San Francisco, C A 94143, IVERSEN L. (1975) High affinity uptake of neurotransmitter USA. amino acids. Narure 253, 481. LEV] G., BERLOTTINI A,, CHENJ. & RAITERI M. (1974) REFERENCES Regional differences in the synaptosomal uptake of 'H-y-aminobutyric acid and '*C-glutamate and possible BENNETT J. P., JR., LOGANW. J. & SNYDER S. H. (1973) role of exchange processes. J . Pharmac. exp. Ther. 188, Amino acids as central nervous transmitters. The in429-438. flucnce of ions, amino acid analogues, and ontogeny on transport systems for L-glutamic and L-aspartic acids LEVIG. & RAITERIM. (1975) Reply. Nature 253, 481-482. 0. H., ROSERROUGHN. J., FARR A. L. & RANDALL and glycine into central nervous synaptosomes of the LOWRY R. J. (1951) Protein measurement with the Fohn phenol rat. J . Neurochem 21, 1533-1550. reagent. J . biol. Chem. 193, 265-275. A. (1965) The isolation and EARLD. C. N. & KORMER L. L. (1969) Subcellular distribution properties of cardiac ribosomes and polysomes. Biochern. NEALM. J. & IVERSEN of endogenous and L3H]-y-aminobutyric acid from brain J . 94, 721-734. slices. J. Neuroc!ient. 16, 1245-1252. GELRART A. & GOLDMAN R. (1977) Correlation between ROSKOSKI R. (1977) Net uptake of L-glutamate and GABA microsomal Na*, K + ATPase activity and L'H]ouabain by high affinity synaptosomal transport systems. Soc. binding to heart tissue homogenates. Biochitn. biophys. Neurosci. Abstr. 7, 1315. Acta 481, 689494. J. S. (1974) Autoradiographic localizaHARRIS W. E., SWANSON P. D. & STAHLW. L. (1973) Oua- SCHONF. & KELLEY tion of 13H]GABA and L3H]glutamate over satellite bain binding sites and the (Na+, K+)-ATPase of brain glial cells. Brain Rex 66, 275-278. microsomal membranes. Biochini. biophys. Acta 298, SNYDER S. H., KUHARM. J., GREENA. I., COYLEJ. T. 68M89. & SHASKAN E. G. (1970) Uptake and subcellular localiHITZEMANN R. J. & LOH H. H. (1978~)A comparison of zation of neurotransmitters in the brain. Inr. Rev. NeuroGABA and 8-alanine transport and GABA membrane bid. 13, 127-158. binding in the rat brain. J. Neurochem. 30, 471477. G . L., KIJYLENSTEIRNA B., ERNSTER L. & BERG R. J. & LOH H. H. (197%) Effects of some SOTTOCASA HITZEMANN STAND A. (1974) An electron transport system associated conformationally restricted GABA analogues on GABA membrane binding and nerve ending transport. Brain with the outer membrane of liver mitochondria. J . Cell. B i d . 32, 41 5-438. R ~ s 144, . 163-169. M. J. & SNVDER S. H. (1971) A HITZEMANN R. J. & LOH H. H. High affinity GABA WOFSEYA. R., KCJHAR unique synaptosomal fraction which accumulates glutaand glutamate transport in developing nerve ending parmic and aspartic acids in brain tissue. Proc. narn. Acad. ticles. Brain Res., in press. Sci., U.S.A. 68, 1102-1106. A. (1970) HOKFELDTT., JONSON G. & LYUNGUAHL typing assistance of KAYEWELCH.This study was supported in part by N.I.N.C.D.S. Grant NS-13398.
