Brain Research, 102 (1976) 351-354 ~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
351
Glutamate breakdown during electric field stimulation
RICHARD H. HASCHKE AND JAMES E. HEAVNER ,4nesthesia Research Center and Departments Of Anesthesiology ( R.H.H. and J.E.H.) and Biochemistry ( R.H.H.), School of Medicine, University of Washington, Seattle, Wash. 98195 (U.S.A.)
(Accepted October 15th, 1975)
Several lines of evidence indicate that glutamic acid is a neurotransmitter in the mammalian central nervous system (CNS). For instance, the glutamate concentration profile in the spinal cord fits the expected distribution of the excitatory transmitter released from dorsal root fibers 4 and glutamate is a potent excitant in the spinal cord 2. It has been shown that tritiated glutamate is taken up by CNS tissue and subsequent exposure to depolarizing electric current or high K ÷ concentration results in substantial release of radioactivity6, 8. Possible alterations of this amino acid due to metabolism during incubation were investigated by ion exchange and paper chromatography 9. It was reported that no significant breakdown of glutamic acid (except for some glutamine formation) was observed. Other investigators have reported from less than 10 ~ metabolism of exogenous glutamate 1 to nearly 100 ~ conversion to glutamine 10. The extent of glutamate metabolism in vitro is evidently dependent on such factors as origin of the neural tissue and concentration of glucose in the medium 11. In this report, evidence is presented showing that under our conditions about 15 ~ of exogenous glutamate is converted to glutamine by cat spinal cord slices and 10-23 ~o is converted to two other metabolites. But more importantly, a substantial amount of glutamate is electrochemically degraded by the depolarizing current. This breakdown may lead to erroneous interpretation of the significance of electrically induced release of glutamate from tissue. Thin cross-sections (approx. 0.5 ram) of cat spinal cord were obtained from the lumbar enlargement and placed in oxygenated (95 ~ 02-5 ~oCOe) modified KrebsHenseleit solution 5 at 0 °C. All of the succeeding operations were performed in this medium. A hemisection was incubated in 1 ml of 10-5 M [3-3H]glutamate (1 × 104 mCi/ mmole) for 30 rain at 30 °C. The hemisection was transferred to a 200 #1 chamber containing coiled platinum electrodes (0.4 mm wire, 5 mm diameter coil) and perfused at 1 ml/min for 15 rain. Electrical field stimulation (10 mA, 5 msec pulses and 100 Hz) was then applied for 2 mix with a constant current stimulus isolation unit. Resistance across the electrodes was approximately 10 k ~ . Perfusate (100 #1) was spotted directly on Whatman 3 MM paper for electrophoretic separation (3000 V, 3 h) in volatile buffers as described by McKenzie and Dawson 7 at various pH values. After the paper
352 TABLE I ELECTROPHORESIS AT
pH
3.2
The origin is at the arrow ; and indicate polarity. Relative positions of standards : ~,-aminobu~yric acid (---30), cz-ketoglutaric acid ( 4 0 ) , 2-pyrrolidone-5-carboxylic acid ( ~ 20). Sample
( ~rd slice
Percentage o f total counts in I cm segments 2
1. 2. 3. 4. 5.
[3-ZH]Glutamate Prestimulation Electrical stimulation Electrical stimulation K + stimulation
no yes yes no yes
14 14 15
- 1 ~ I
98 68* 9* 53 75**
2
3
4
5
2 6 8 22 4
6
7
8
9
54 25
I0
/l
12 15 6
* These peaks were skewed and were shown at pH 4.5 electrophoresis to be about 15 % glutamine. ** Krebs-Henseleit with final K + --- 62.5 mM and Na + == 81.7 raM; total osmolarity is unchanged.
was dried, amino acid standards were detected by ninhydrin, a-Ketoglutarate and 2pyrrolidone-5-carboxylic acid standards were visualized respectively by the methods of Elfron 3, and Zweig and WhitakedL When radioactivity was to be measured, electrophoretically developed but unstained strips were cut into 1 cm segments and the soluble contents o f each segment eluted in the scintillation vial with 500 #1 of H~O. Bray's solution was then added to the vial and radioactivity determined. Before stimulation of the tissue, electrophoretic separation (pH 3.2) of the perfusate from a cord slice incubated in [3-3H]glutamate showed glutamate, glutamine and two unidentified metabolites (Table I, sample 2). Neither of the unidentified compounds comigrated with c~-ketoglutaric acid, 7-aminobutyric acid, 2-pyrrolidone-5carboxylic acid or glutamine. The same perfusate collected during stimulation contained these two unknowns and a third also derived from glutamate (sample 3). Electrophoresis at pH 4.7 clearly separated the glutamate and glutamine peaks and showed that glutamine was unaltered by tissue stimulation. Current passage through 1/zM glutamate in the absence of a cord slice (sample 4) increased the amount of contaminant present in the original [3-~H]glutamate (sample 1) and apparently produced the same unknown formed when tissue was present (sample 3). Subsequent experiments demonstrated that a constant quantity of glutamate (approx. 10-6 M) was degraded regardless of its concentration (10-6-10 -3 M), suggesting saturation of a catalytic surface. Electrolysis of glutamate was identical with H20 (with sufficient NaC1 for current flow), acid, base or Krebs-Henseleit solutions and occurred whether platinum, gold, stainless steel, carbon or NaCl/paper wick electrodes were used. In contrast to electric field stimulation, K + depolarization did not cause the conversion of glutamate (Table I, sample 5). Electrophoretic separation of a mixture of [1-14C]- and [3-3H]glutamate following electrical field stimulation in the absence of tissue (Fig. 1, stimulated) demonstrated that the electrochemical breakdown products found with the [3-ZH]glutamate
353
BO
,' J ,'
20
10
r I
I'LI
r
II',
r
iI
3H ( C ' 3 ) STIMULATED
",
I i' I II
L I I
I I I
II
',t'L
I
\ k\ 14 C(C~1/
0 × ~r a.
15
UNSTIMULAT};D
A
i/
3H
(C-3)
/ J "~''~ ¢ / I
CD /
-3
-1 1
3
~1
5
7 9 SEGMENT
14C~ C )
11
"3
15
"7
Fig. 1. Electrophoretic profile of [3-SH]glutamate and [I-uC]glutamate at pH 3.2. Each amino acid was 1 x 10-6 M in 1.5 mM NaC1. Stimulation was for 2 min under standard conditions. were not observed when [1-14C]glutamate was used. This indicated that the a-carboxyl group (C-l) had been removed from the glutamate molecule. When the electrophoretic separation of the stimulated [3-SH]glutamate was carried out at pH 1.9 (a-carboxyl group 6 0 ~ charged, pK 2.10, and 7-carboxyl group < 1 ~ charged, pK 4.07; amino group 100K positively charged, pK 9.47), unaltered glutamate migrated toward the negative electrode as expected, The breakdown product did not move from the origin which suggested the absence of the positively charged a-amino group (7-carboxyl uncharged). A plausible explanation would be that glutamate is decarboxylated and deaminated in an electrochemical reaction resulting in a 4-carbon carboxylic acid. Positive chemical identification of the substance formed by the electrolysis would be difficult because, even though a high percentage of the glutamate is degraded, the quantity generated is extremely small relative to what is required for chemical analysis. Since the product formed by electrochemical breakdown of glutamate is not produced
354 by the tissue, it is unlikely that it is b i o l o g i c a l l y significant. T h e i m p o r t a n t p o i n t is n o t the e x a c t c h e m i c a l s t r u c t u r e o f the b r e a k d o w n p r o d u c t , but that such d e c o m p o s i t i o n m a y likely o c c u r u n d e r c o n d i t i o n s o f tissue s t i m u l a t i o n f r e q u e n t l y used to i n d u c e g l u t a m a t e release. S u p p o r t e d by N I H A n e s t h e s i a R e s e a r c h C e n t e r G r a n t G M - 1 5 9 9 1 .
l BALCAR, V. J., AND JOHNSTON, G. A. R., High affinity uptake of transmitters: studies on the uptake
2 3 4
5 6 7
8 9 10 11
12
of L-aspartate, GABA, L-glutamate, and glycine in cat spinal cord, J. Neurachem., 20 (1972) 529-539. CURTIS, D. R., PHILLIS, J. W., AND WATKINS, J. C., The chemical excitation of spinal neurons by certain acidic amino acids, J. Physiol. (Lond.), 150 (1960) 656-682. ELFRON, M, L,, High voltage paper electrophoresis, in I. SMm4 (Ed.), Chromatographic' and Electrophoretic Techniques, Vol. II, 2nd ed., Interscience, New York, 1960, p. 191. GRAHAM,L. T., SHANK~R. P., WERMAN, R., AND APRISON, M. H., Distribution of some synaptic transmitter suspects in cat spinal cord : glutamic acid, asparatic acid, 7-aminobutyric acid, glycine, and glutamine, J. Neurochem., 14 (1967) 465-472. HEAVNER,J. E., AND DEJONG, R. H., Lidocaine blocking concentrations for B- and C-nerve fibers, Anesthesiology, 40 (1974) 228-233. HOPKIN, J., AND NEAL, M. J., Effect of electrical stimulation and high potassium concentrations on the efflux of [14C]glycine from slices of spinal cord, Brit. J. Pkarmacol., 42 (1971) 215-223. MCKENZIE, H. A., AND DAWSON, R. M. C., pH and buffers and physiological media, In R. M. C. DAWSON, D. C. ELLIOTT, W. H. ELLIOTTAND K. M. JONES(Eds.), Data for Biochemical Research, 2nd ed., Oxford University Press, New York, 1969, p. 506. MULOER, A. H., AND SNVDER, S. H., Potassium-induced release of amino acids from cerebral cortex and spinal cord slices of the rat, Brain Research, 76 (1974) 297-308. ROBERTS,P. H., AND MITCHELL,J. F., The release of amino acids from the hemisected spinal cord during stimulation, J. Neurochem., 19 (I 972) 2473-2481. SHANK, R. P., WHITEN, J. T., AND BAXTER,C. F., Glutamate uptake by the isolated toad brain, Science, 181 (1973) 860 862. WEIL-MALHERBE,H., AND GORDON, J., Amino acid metabolism and ammonia formation in the brain slices, J. Neurochem., 18 (1971) 1659-1672. ZWEIC, G., AND WH~TAKER,J. R., Organic acids and derivatives. In J. R. WHITAKER(Ed.), Paper Chromatography and Electrophoresis, Vol. I, Academic Press, New York, 1967, p. 288.
351
Glutamate breakdown during electric field stimulation
RICHARD H. HASCHKE AND JAMES E. HEAVNER ,4nesthesia Research Center and Departments Of Anesthesiology ( R.H.H. and J.E.H.) and Biochemistry ( R.H.H.), School of Medicine, University of Washington, Seattle, Wash. 98195 (U.S.A.)
(Accepted October 15th, 1975)
Several lines of evidence indicate that glutamic acid is a neurotransmitter in the mammalian central nervous system (CNS). For instance, the glutamate concentration profile in the spinal cord fits the expected distribution of the excitatory transmitter released from dorsal root fibers 4 and glutamate is a potent excitant in the spinal cord 2. It has been shown that tritiated glutamate is taken up by CNS tissue and subsequent exposure to depolarizing electric current or high K ÷ concentration results in substantial release of radioactivity6, 8. Possible alterations of this amino acid due to metabolism during incubation were investigated by ion exchange and paper chromatography 9. It was reported that no significant breakdown of glutamic acid (except for some glutamine formation) was observed. Other investigators have reported from less than 10 ~ metabolism of exogenous glutamate 1 to nearly 100 ~ conversion to glutamine 10. The extent of glutamate metabolism in vitro is evidently dependent on such factors as origin of the neural tissue and concentration of glucose in the medium 11. In this report, evidence is presented showing that under our conditions about 15 ~ of exogenous glutamate is converted to glutamine by cat spinal cord slices and 10-23 ~o is converted to two other metabolites. But more importantly, a substantial amount of glutamate is electrochemically degraded by the depolarizing current. This breakdown may lead to erroneous interpretation of the significance of electrically induced release of glutamate from tissue. Thin cross-sections (approx. 0.5 ram) of cat spinal cord were obtained from the lumbar enlargement and placed in oxygenated (95 ~ 02-5 ~oCOe) modified KrebsHenseleit solution 5 at 0 °C. All of the succeeding operations were performed in this medium. A hemisection was incubated in 1 ml of 10-5 M [3-3H]glutamate (1 × 104 mCi/ mmole) for 30 rain at 30 °C. The hemisection was transferred to a 200 #1 chamber containing coiled platinum electrodes (0.4 mm wire, 5 mm diameter coil) and perfused at 1 ml/min for 15 rain. Electrical field stimulation (10 mA, 5 msec pulses and 100 Hz) was then applied for 2 mix with a constant current stimulus isolation unit. Resistance across the electrodes was approximately 10 k ~ . Perfusate (100 #1) was spotted directly on Whatman 3 MM paper for electrophoretic separation (3000 V, 3 h) in volatile buffers as described by McKenzie and Dawson 7 at various pH values. After the paper
352 TABLE I ELECTROPHORESIS AT
pH
3.2
The origin is at the arrow ; and indicate polarity. Relative positions of standards : ~,-aminobu~yric acid (---30), cz-ketoglutaric acid ( 4 0 ) , 2-pyrrolidone-5-carboxylic acid ( ~ 20). Sample
( ~rd slice
Percentage o f total counts in I cm segments 2
1. 2. 3. 4. 5.
[3-ZH]Glutamate Prestimulation Electrical stimulation Electrical stimulation K + stimulation
no yes yes no yes
14 14 15
- 1 ~ I
98 68* 9* 53 75**
2
3
4
5
2 6 8 22 4
6
7
8
9
54 25
I0
/l
12 15 6
* These peaks were skewed and were shown at pH 4.5 electrophoresis to be about 15 % glutamine. ** Krebs-Henseleit with final K + --- 62.5 mM and Na + == 81.7 raM; total osmolarity is unchanged.
was dried, amino acid standards were detected by ninhydrin, a-Ketoglutarate and 2pyrrolidone-5-carboxylic acid standards were visualized respectively by the methods of Elfron 3, and Zweig and WhitakedL When radioactivity was to be measured, electrophoretically developed but unstained strips were cut into 1 cm segments and the soluble contents o f each segment eluted in the scintillation vial with 500 #1 of H~O. Bray's solution was then added to the vial and radioactivity determined. Before stimulation of the tissue, electrophoretic separation (pH 3.2) of the perfusate from a cord slice incubated in [3-3H]glutamate showed glutamate, glutamine and two unidentified metabolites (Table I, sample 2). Neither of the unidentified compounds comigrated with c~-ketoglutaric acid, 7-aminobutyric acid, 2-pyrrolidone-5carboxylic acid or glutamine. The same perfusate collected during stimulation contained these two unknowns and a third also derived from glutamate (sample 3). Electrophoresis at pH 4.7 clearly separated the glutamate and glutamine peaks and showed that glutamine was unaltered by tissue stimulation. Current passage through 1/zM glutamate in the absence of a cord slice (sample 4) increased the amount of contaminant present in the original [3-~H]glutamate (sample 1) and apparently produced the same unknown formed when tissue was present (sample 3). Subsequent experiments demonstrated that a constant quantity of glutamate (approx. 10-6 M) was degraded regardless of its concentration (10-6-10 -3 M), suggesting saturation of a catalytic surface. Electrolysis of glutamate was identical with H20 (with sufficient NaC1 for current flow), acid, base or Krebs-Henseleit solutions and occurred whether platinum, gold, stainless steel, carbon or NaCl/paper wick electrodes were used. In contrast to electric field stimulation, K + depolarization did not cause the conversion of glutamate (Table I, sample 5). Electrophoretic separation of a mixture of [1-14C]- and [3-3H]glutamate following electrical field stimulation in the absence of tissue (Fig. 1, stimulated) demonstrated that the electrochemical breakdown products found with the [3-ZH]glutamate
353
BO
,' J ,'
20
10
r I
I'LI
r
II',
r
iI
3H ( C ' 3 ) STIMULATED
",
I i' I II
L I I
I I I
II
',t'L
I
\ k\ 14 C(C~1/
0 × ~r a.
15
UNSTIMULAT};D
A
i/
3H
(C-3)
/ J "~''~ ¢ / I
CD /
-3
-1 1
3
~1
5
7 9 SEGMENT
14C~ C )
11
"3
15
"7
Fig. 1. Electrophoretic profile of [3-SH]glutamate and [I-uC]glutamate at pH 3.2. Each amino acid was 1 x 10-6 M in 1.5 mM NaC1. Stimulation was for 2 min under standard conditions. were not observed when [1-14C]glutamate was used. This indicated that the a-carboxyl group (C-l) had been removed from the glutamate molecule. When the electrophoretic separation of the stimulated [3-SH]glutamate was carried out at pH 1.9 (a-carboxyl group 6 0 ~ charged, pK 2.10, and 7-carboxyl group < 1 ~ charged, pK 4.07; amino group 100K positively charged, pK 9.47), unaltered glutamate migrated toward the negative electrode as expected, The breakdown product did not move from the origin which suggested the absence of the positively charged a-amino group (7-carboxyl uncharged). A plausible explanation would be that glutamate is decarboxylated and deaminated in an electrochemical reaction resulting in a 4-carbon carboxylic acid. Positive chemical identification of the substance formed by the electrolysis would be difficult because, even though a high percentage of the glutamate is degraded, the quantity generated is extremely small relative to what is required for chemical analysis. Since the product formed by electrochemical breakdown of glutamate is not produced
354 by the tissue, it is unlikely that it is b i o l o g i c a l l y significant. T h e i m p o r t a n t p o i n t is n o t the e x a c t c h e m i c a l s t r u c t u r e o f the b r e a k d o w n p r o d u c t , but that such d e c o m p o s i t i o n m a y likely o c c u r u n d e r c o n d i t i o n s o f tissue s t i m u l a t i o n f r e q u e n t l y used to i n d u c e g l u t a m a t e release. S u p p o r t e d by N I H A n e s t h e s i a R e s e a r c h C e n t e r G r a n t G M - 1 5 9 9 1 .
l BALCAR, V. J., AND JOHNSTON, G. A. R., High affinity uptake of transmitters: studies on the uptake
2 3 4
5 6 7
8 9 10 11
12
of L-aspartate, GABA, L-glutamate, and glycine in cat spinal cord, J. Neurachem., 20 (1972) 529-539. CURTIS, D. R., PHILLIS, J. W., AND WATKINS, J. C., The chemical excitation of spinal neurons by certain acidic amino acids, J. Physiol. (Lond.), 150 (1960) 656-682. ELFRON, M, L,, High voltage paper electrophoresis, in I. SMm4 (Ed.), Chromatographic' and Electrophoretic Techniques, Vol. II, 2nd ed., Interscience, New York, 1960, p. 191. GRAHAM,L. T., SHANK~R. P., WERMAN, R., AND APRISON, M. H., Distribution of some synaptic transmitter suspects in cat spinal cord : glutamic acid, asparatic acid, 7-aminobutyric acid, glycine, and glutamine, J. Neurochem., 14 (1967) 465-472. HEAVNER,J. E., AND DEJONG, R. H., Lidocaine blocking concentrations for B- and C-nerve fibers, Anesthesiology, 40 (1974) 228-233. HOPKIN, J., AND NEAL, M. J., Effect of electrical stimulation and high potassium concentrations on the efflux of [14C]glycine from slices of spinal cord, Brit. J. Pkarmacol., 42 (1971) 215-223. MCKENZIE, H. A., AND DAWSON, R. M. C., pH and buffers and physiological media, In R. M. C. DAWSON, D. C. ELLIOTT, W. H. ELLIOTTAND K. M. JONES(Eds.), Data for Biochemical Research, 2nd ed., Oxford University Press, New York, 1969, p. 506. MULOER, A. H., AND SNVDER, S. H., Potassium-induced release of amino acids from cerebral cortex and spinal cord slices of the rat, Brain Research, 76 (1974) 297-308. ROBERTS,P. H., AND MITCHELL,J. F., The release of amino acids from the hemisected spinal cord during stimulation, J. Neurochem., 19 (I 972) 2473-2481. SHANK, R. P., WHITEN, J. T., AND BAXTER,C. F., Glutamate uptake by the isolated toad brain, Science, 181 (1973) 860 862. WEIL-MALHERBE,H., AND GORDON, J., Amino acid metabolism and ammonia formation in the brain slices, J. Neurochem., 18 (1971) 1659-1672. ZWEIC, G., AND WH~TAKER,J. R., Organic acids and derivatives. In J. R. WHITAKER(Ed.), Paper Chromatography and Electrophoresis, Vol. I, Academic Press, New York, 1967, p. 288.
