Brain Research, 143 (1978) 487-498 © Elsevier/North-HollandBiomedicalPress
487
GLYCINE HIGH AFFINITY UPTAKE AND STRYCHNINE BINDING ASSOCIATED WITH GLYCINE RECEPTORS IN THE FROG CENTRAL NERVOUS SYSTEM
WALTER E. MOLLER and SOLOMON H. SNYDER* Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Md. 21205 (U.S.A.)
(Accepted July 14th, 1977)
SUMMARY Accumulation of [aH]glycine into synaptosomal fractions occurs by high affinity systems in cerebral cortex, optic tectum, brain stem and spinal cord of the frog. Specific [all]strychnine binding which appears associated with postsynaptic glycine receptors is also demonstrable in these regions. By contrast, only very low levels of strychnine binding and high affinity glycine uptake occur in higher centers of the rat central nervous system. The relative potencies of small neutral amino acids in competing for [SH]strychnine binding are similar in frog brain and spinal cord. No evidence for a high affinity accumulation of [3H]taurine by synaptosomal fractions of frog spinal cord can be demonstrated. These observations favor glycine rather than taurine as an inhibitory transmitter in frog spinal cord. Moreover, these findings suggest that glycine may have a synaptic role in higher brain centers in the frog.
INTRODUCTION A variety of neurophysiological and neurochemical evidence indicates that glycine is a major inhibitory neurotransmitter in the spinal cord and brain stem of the mammalian central nervous system. Both natural postsynaptic inhibition and the effects of glycine in spinal cord and brain stem are blocked selectively by strychnineS, s, 9,27,2s. High affinity, sodium-dependent uptake systems for glycine into unique synaptosomal fractions have been demonstrated in mammalian spinal cord and brain stem 2,1s,21. Accumulated [aH]glycine is released selectively by depolarization of spinal cord and brain stem slices ~a. Endogenous levels of glycine are highest in spinal cord * To whom reprint requests should be addressed.
488 and brain stem1,15. Sites associated with glycine receptors can be labeled in mammalian spinal cord and brain stem by the binding of [3H]strychnine31,32. While neurochemical and neurophysiological studies indicate unique synaptic actions of glycine in mammalian spinal cord and brain stem, glycine appears to lack such properties in mammalian higher centers such as the cerebral cortex. Strychninesensitive hyperpolarization of neurons is not readily demonstrated in mammalian cerebral cortex3,19,2°. The high affinity glycine uptake system into unique synaptosomal fractions appears to be absent in mammalian cerebral cortex2,1s,21. Only negligible depolarization-induced release of accumulated [aH]glycine can be demonstrated from rat cerebral cortex slices23. Mammalian cerebral cortex also appears to lack detectable strychnine binding associated with glycine receptors 31. Recent evidence suggests that in the frog glycinergic elements may play a major role in higher centers. High affinity, sodium-dependent accumulation of glycine into cerebral cortex slices of the frog has been demonstrated1°. Potassium depolarization releases accumulated [aH]glycine from slices of frog cerebral cortex 1~. Neurophysiological evidence suggests that taurine rather than glycine may mediate strychnine-blocked postsynaptic inhibition in the frog spinal cord. Taurine is more potent than glycine in hyperpolarizing frog motor neurons ~4,26. Moreover, the inhibitory effects of taurine are more sensitive to blockade by strychnine than are those of glycine ~6. Endogenous levels of taurine in frog spinal cord are substantially higher than those of glycine 7. To clarify the possible synaptic role of glycine and other neutral amino acids such as taurine in the frog central nervous system, we have evaluated the accumulation of [aH]glycine by synaptosomal fractions as well as apparent receptor binding of [all]strychnine. METHODS
Animals Bullfrogs (Rana catesbiana, 6"-8" body length), chicken (3 weeks old), and male Sprague-Dawley rats were used. The frogs were obtained from Mogul-ED Corporation of Oshkosh, Wisconsin.
Membrane preparation The animals were decapitated and the brains and spinal cords rapidly removed. Frog and rat brains were dissected by the methods of Yates and Taberner 29 or Glowinski and Iversen14 respectively. Synaptic membranes were prepared as previously described for rat brain la and rat spinal cord 31,az. The final membrane pellet was frozen at --20 °C. Protein was measured by the method of Lowry et al.2L
[3H]strychnine binding The binding of [all]strychnine to synaptic membranes was determined by a modification6 of a previously described procedure al,32 in order to economize on tissue. The membrane pellet was homogenized in ice-cold 0.05 M sodium-potassium
489 phosphate buffer containing 200 mM NaC1 at pH 7.1 s2, using a Brinkmann Polytron PT-10. For the standard binding assay, 1-ml aliquots of tissue suspension (about 0.4 mg protein/ml) were added to test tubes containing Jail]strychnine (final concentration about 6 nM) and various amounts of non-radioactive strychnine or other compounds. After an incubation at 4 °C for 10 min a2, three 250-#1 aliquots of each incubation mixture were removed to plastic microfuge tubes (Beckman, 0.4 ml capacity) and centrifuged for 2.5 rain in a Beckman Microfuge B. The supernatant was removed by aspiration and each pellet rinsed superficially once with 250/A ice-cold incubation buffer. The bottom of each microfuge tube was cut off into a scintillation vial; the pellets were dissolved in 1 ml Protosol (New England Nuclear) and radioactivity was determined in 10 ml LSC (Yorktown Research) by liquid scintillation spectrometry at a counting efficiency of 43 ~. Specific [3H]strychnine binding was obtained by subtracting from the total bound radioactivity the amount not displaced by high concentrations of strychnine (0.1 mM) or glycine (1 mM). Values for specific binding of [all]strychnine were the same whether strychnine or glycine was used as displacer.
Uptake studies Uptake of amino acids by nuclei-free 1:20 sucrose (0.32 M) homogenates was assayed as previously described 21. Aliquots (0.2 ml) of the homogenates were incubated for 4 min at 37 °C together with 2.8 ml of Krebs-Ringer-Tris.HCl (pH 7.4 at 37 °C), containing various concentrations of the radioactive amino acids. Incubation mixtures were rapidly cooled and centrifuged at 28,000 × g for 10 min. The supernatant fluid was discarded and the pellets washed once with ice-cold 0.15 M NaC1 solution, resuspended in 10 ml ice-cold 0.15 M NaCI, and recentrifuged at 48,000 × g for 10 min. The final pellets were dissolved in 1 ml Protosol and the radioactivity was determined in 10 ml LSC by liquid scintillation spectrometry. Under these conditions [3H]glycine is accumulated primarily into synaptosomal fractions of rat 2,21 and frog spinal cord (W. Miiller, in preparation).
Materials [all]strychnine (radioactive purity 90~, specific activity 8.7 Ci/mmole) was labelled by catalytic tritium exchange as described previouslyal. [aH]glycine (11 Ci/mmole) and [3H]taurine (18 Ci/mmole) were obtained from New England Nuclear and Amersham/Searle respectively. All other substances were obtained from commercial suppliers. RESULTS
Saturation of [3H]glycine uptake and [all]strychnine binding [SH]glycine is accumulated by synaptosomal fractions of frog brain and frog spinal cord in a saturable fashion (Fig. 1). Both in brain and spinal cord discrete high and low affinity systems can be demonstrated as distinct components in repeated experiments. The Km values (25-30/~M) for the high affinity uptake system are similar in brain and spinal cord and resemble values for high affinity glycine uptake
490
I0 -
Fro~ Broi__n
HighAffinity System 0.8 Km =24 8_+[.4~M Vmax=455 4-025 / •
I/v o~ •
~,~ / -
/~..~/•__/
LOW Affinity System
/l~'e/Km" 02 ~" 4"~ i
i
IC •
389 -+I)~M Vmox=24,85:i:2.21
r
i
i
i
i
Frog Spinol Cord
HighAfhnlty System 08 Km=290_+07ffM Vmex=608+014 /
•
OA e~
•
LOW Affinity System
02 ~ " - /
Km=292±24/'~M
4
Vme*~)5.18 ±0.57
~
/ , 0,2
O'6
1 0.4
0 r8
' 1.0
' 12
I
",5 ~[OLYC,NE] .,0 M
Fig. 1. Double reciprocal plot of pH]glycine uptake in frog brain and frog spinal cord homogenates. Uptake of various concentrations of [aH]glycine was measured as described in Methods. In each case the plot could be resolved into 2 straight-line components in 4 repeated experiments. The Km and Vm a* values reported are means ~: S.E.M. of 4 determinations. Velocity (V) is expressed as m o l e s per mg protein/4 min.
0_
6
bJ
o
•
specific
/
~02
o nonspecific ,=,
4
a z o m hl z z -to >-
r-~
FROG SPINAL C
o
O
R
D
~
Total Binding Sites:2.4 pmole/rng prot
~o.-,,
~ 0,1 >-
• ~ -
(0 I
r-~ i
i
i
i
i
20 40 60 [3H]-STRYC HN, NE
i
~
0 ~M]
i
i
I00
nM
"."X,,.
2
i
KD:II
I
,\
2
[SH]-STRYCHNINE BOUND[nM]
L
3
Fig. 2. Saturation of specific [all]strychnine binding to frog spinal cord membranes, pH]strychnine binding was determined using the standard assay described in Methods with increasing concentrations of [all]strychnine. Non-sp~ific binding was obtained from experiments conducted in the presence of 0.1 m M strychnine or 1 m M glycine. The points are means of thr¢~ det~minations which varied less than 20%. The experiment was replicated 3 times. Left: linear plot. Right: Scatchard analysis of specific pH]strychnine binding.
491
o
4
:~' "6
3
• specific o nonspecific
E ,m z
FROG BRAIN 2
o
"r
F-
'
20
40
60
80
I00
[3H].STRYCHNINE [n~ ~ 005 Z 0 004 °
Z 003
Total Binding Sites, lO pmole/mg protein KD:I6nM
L.\
~oo2
\
I--
•
,~ oo,
\ • I
I 2
I 3
p.]-s-r..,c..,.E BouNo[ 4 Fig. 3. Saturation of specific [all]strychnine binding to frog whole brain membranes. Experimental conditions were the same as in Fig. 2. Upper part: linear plot. Lower part: Scatcbard analysis of specific [all]strychnine binding. The experiment was replicated 3 times.
previously reported in mammalian spinal cord synaptosomal fractions 21. The calculated maximal number of uptake sites for the high affinity system is slightly higher in the spinal cord than the brain stem. Both Km and Vmax values for low affinity accumulation of glycine are similar in frog brain and spinal cord. Saturable binding of [all]strychnine is demonstrable in crude synaptic membrane preparations of both frog spinal cord and whole frog brain (Figs. 2 and 3). At 6 nM [all]strychnine, the concentration used in routine binding studies, total [all]strychnine binding is about 3 and 2 times higher than the values for non-specific binding taken in the presence of 1 mM glycine or 0.1 mM non-radioactive strychnine in spinal cord and brain respectively. In both spinal cord and brain membranes, nonspecific binding values increase linearly with [all]strychnine concentration. By contrast, specific binding plateaus at about 20 nM [all]strychnine with half-maximal values at about 8-12 nM. Dissociation constants (KD) calculated for [all]strychnine binding in frog spinal cord and brain are 11 nM and 16 riM, respectively.
492 TABLE I Regional distribution of high affinity [aH Jgb,cine uptake and [ 3H ]strychnine binding in frog and rat C N S
High affinity [3H]glycine uptake was determined using 5 different [aH]glycine concentrations over the range of 8-30 uM. [3H]strychnine binding was determined by the assay procedure described in Methods, using a [all]strychnine concentration of 6 nM. Values reported are means ± S.E.M. of 4 (uptake) or 5 (Jail]strychnine binding) determinations. Glycine uptake values for rat cerebral cortex are in parentheses, because accumulation in this area does not appear to involve a high affinity system. High affinity [3H]glycine uptake
: 3Hjstrychnine binding
Km ~: (10 5 M)
V. . . . (nmole/rng protein~4")
(pmole/mg protein)
Frog Spinal cord Brain stem Optic tectum Cerebral cortex
2.90 4.13 4.05 3.60
± ± ± ±
0.07 0.14 0.37 0.32
6.08 3.88 3.00 3.07
± -i ±
0.14 0.25* 0.25* 0.20",**
0.80 0.33 0.21 0.10
~ i ± £
Rat Spinal cord Brain stem Midbrain Cerebral cortex
4.30 4.58 4.72 (43.30
± i ± ±
0.14 0.17 0.13 5.5)
5.31 ~ 2.91 ± 1.78 ± (9.03 ±
0.10 0.02* 0.08",** 1.38)
0.54 0.45 0.13 0.01
± 0.03 ± 0.02* ± 0.01",** j_: 0.003",**
0.03 0.01" 0.01",** 0.10",**
* When compared with the spinal cord, P < 0.001. ** When compared with the brain stem, P < 0.05.
Regional variations in [3H]glyeine uptake and [ 3Hjstrychnine binding The substantial levels of high affinity glycine uptake and of [3H]strychnine binding in whole frog brain contrasts with the very low levels of both in rat brain. Accordingly, we c o m p a r e d high affinity glycine uptake and [3H]strychnine binding in different regions of frog brain (Table I). Saturable accumulation of [ZH]glycine and [3H]strychnine binding are demonstrable in frog cerebrum, optic tectum, brain stem and spinal cord. The Km value for glycine accumulation is similar in the 4 areas. The maximal n u m b e r of glycine accumulating sites is highest in the spinal cord with values in the brain stem being a b o u t 64 ~ of spinal cord levels and densities in the optic rectum and cerebral cortex, cortex being about half o f spinal cord levels. Specific [ZH]strychnine binding is also highest in the spinal cord, about 2.4 times higher than the next highest levels which occur in the brain stem. Strychnine binding levels in the optic tectum and cerebral cortex are one-quarter and one-eighth, respectively, o f values in the spinal cord. These findings differ markedly from results in the rat cerebral cortex, where no high affinity uptake of glycine can be demonstrated (Table I) 18,21. Moreover, in the rat only negligible amounts o f [all]strychnine binding can be demonstrated in the cerebral cortex (Table I). In the rat, as in the frog ,the brain stem does possess substantial levels of high affinity glycine accumulation and [all]strychnine binding (Table I).
493 TABLE II Glycine uptake and [all]strychnine binding in brain and spinal cord of rat, chicken andfrog
[aH]glycine uptake was determined using 10 different [aH]glycineconcentrations over the range of 8-1000/zM. Values are means 4- S.E.M. of 4 determinations. [all]strychnine binding was determined by the standard procedure described in Methods, using 8 different [all]strychnine concentrations as shown in Figs. 2 and 3. The total number of binding sites and the Ko was determined by Scatchard analysis (Figs. 2 and 3). Values are means of three determinations which varied less than 20 %. [ZH]glycine uptake High affinity system Km (10-5 M)
Rat brain Chicken brain Frog brain Rat spinal cord Chicken spinal cord Frog spinal cord
Low affinity system
Vmax Km (Izmole/mg (10 4 M) protein/4")
{ZH]strychnine binding
Maximal KD I'max binding (nM) (l~m°le/mg (pmole/mg protein~4') protein)
3.40±0.13 1.934-0.28 7.564-1.4 19.14-3.6
High affinity glycine uptake
Vmnx
Maximal strychnine binding
0.3
9
6.4
3.7010.20 1.294-0.04 7.845:0.7 11.94-1.1 0.2 2.484-0.14 4.55±0.23 3.194-0.1 24.84-2.2 1.0
12 16
6.5 4.6
4.654-0.16 5.705:0.07 4.434-0.3 20.14-0.8 1.9
13
3.0
4.184-0.10 2.604-0.07 6.694-0.4 11.24-1.1
0.95
16
2.7
2.904-0.07 6.084-0.14 2.924-0.2 15.14-0.5
2.4
11
2.5
Comparison o f [3H]glycine uptake and [all]strychnine binding in chicken, rat and frog
Because of the differences in the properties of glycine uptake and receptor binding in frog and rat, we sought to evaluate a different animal class. Accordingly, in parallel experiments we compared properties of accumulation of [aH]glycine and binding of [all]strychnine in spinal cord and whole brain of chicken, frog and rat (Table II). Both high and low affinity systems for [SH]glycine uptake are apparent in whole brain and spinal cord of all three species. Km values for high affinity [aH]glycine uptake are fairly similar in all species examined and in both brain and spinal cord. Differences among the species are apparent, however, in the ratio of the Vmax values for glycine accumulation in spinal cord and brain. In the rat the maximal number of uptake sites in the spinal cord is triple values for whole rat brain. In the chicken the spinal cord Vmax is about double the value in brain, while, in the frog, Vmax values are only 1.3 times higher in spinal cord than brain. In contrast to the marked differences among the three species in high affinity glycine accumulation in spinal cord and brain, Vmax values for low affinity glycine uptake do not markedly differentiate the three species. For rat, chicken and frog, the amount of low affinity glycine accumulation is similar in spinal cord and brain. The KD values for [3H]strychnine binding in brain and spinal cord of the three species are quite similar (Table II). In all three species the number of strychnine binding sites is greater in spinal cord than in whole brain. However, there are species differences in the ratio of spinal cord to whole brain. In the rat, maximal [3H]-
494 strychnine binding is 6.3 times higher in spinal cord than brain and in the chicken the corresponding ratio is 4.7. By contrast, in the frog, the number of spinal cord binding sites in only twice the number of whole brain binding sites. Relative amounts of high affinity glycine uptake and [all]strychnine binding display similarities among the three species. Thus, while the absolute values for the Vmax of high affinity [aH]glycine and [3H]strychnine binding in whole brain of rat, chicken and frog may vary more than 2-3-fold, the ratio of glycine uptake and strychnine binding is quite similar for the three species. In the spinal cord the Vm ~x of the high affinity glycine uptake system and the maximal number of strychnine binding sites vary among species more than two-fold, while the ratio of these values is similar for the three species. Though whole brain levels of both high affinity glycine uptake and strychnine binding are less than for spinal cord in all three species, the difference between brain and spinal cord is greater for strychnine binding than for glycine uptake. The proportionately higher levels of high affinity glycine uptake in whole brain relative to spinal cord might conceivably reflect glycine uptake by constituents other than 'glycinergic' neurons. Several workers have demonstrated high affinity sodiumdependent accumulations of amino acids by glial cells in the central nervous system4,16,~7,25. Substrate specificity o f jail]strychnine binding in ]rog, rat and chicken
Because of neurophysiologic evidence that taurine is more potent than glycine in eliciting synaptic hyperpolarization in the frog spinal cord, we compared the potencies of glycine, taurine and fl-alanine in competing for [all]strychnine binding to spinal cord membranes of chicken, rat and frog (Table III). The affinities ofglycine are fairly similar in spinal cord membranes from the three species with IC50 values of 42-72 #M. In all three species, taurine is substantially less potent than glycine in competing for [all]strychnine binding with IC50 values 2.4-4.4 times greater than those of glycine, flAlanine has about the same affinity as glycine for the strychnine binding sites in all three species. The affinity of strychnine itself is similar in the three species. To ascertain whether strychnine labels the same sites in frog spinal cord and TABLE Ill Substrate specificity of [all]strychnine binding in rat, chicken and frog spinal cord
Inhibition of [SH]strychninebinding to synaptic membranes by amino acids and unlabeled strychnine was determined using the standard procedure described in Methods. IC50values, the concentration of displacer which reduces binding of laH]strychnineby 50 %, were calculated by log-probitanalysis, using 4-5 different concentrations of the displacer. Values are means i S.E.M. of 4 determinations.
Rat Chicken Frog
Glycine IC5o (~M)
Taurine IC5o (I~M)
fl-A lanine 1Cso (I~M)
Strychnine IC~o (nM)
41.7 -4- 6.0 72.3 ± 11.5 55.3 4- 15.1
185.0 4- 2.9 188.3 i 33.7 130.0 4- 8.9
61.3 -4- 7.0 96.7 :~ 24.6 46.7 -l- 6.7
11.3 q- 1.8 14.7 -4- 1.3 10.2 4- 1.4
495 TABLE IV
Substrate specificity of [aHjstrychnine binding in frog CNS IC5o values were determined as described in Table III. Values are means 4- S.E.M. of 4 determinations
Amino acid
Frog brain IC5o (ttM)
Glycine fl-Alanine Taurine L-a-Alanine L-Proline y-Aminobutyric acid L-Glutamic acid Strychnine
33 38 135 > 500 >>500 >>500 >>500 0.01
Frog spinal cord IC5o (#M)
4- 4 ± 4 -4- 17
4- 0.005
55 4- 15 47 :E 7 130 -4- 9 > 500 >>500 >>500 >>500 0.01 4- 0.001
brain, we compared the substrate specificity of [3H]strychnine binding in these two regions (Table IV). The IC50 value of strychnine itself is about the same in whole brain and spinal cord membranes. Of a series of amino acids evaluated, glycine and flalanine have the highest affinities, which is about the same in brain and spinal cord. In both regions the IC50 value for taurine is about 2.4-4.4 times greater than that for glycine. L-a-alanine, L-proline, 7-aminobutyric acid and L-glutamic acid have negligible affinity for strychnine binding sites in frog brain or spinal cord.
Frog Brein 86I/
High Affinity System
Km.66.3.+6~ " - - ~ / " Vmox =12.4-+0.7
2-
f I0
e~"
,/
V 4-
~'~
Low Affini)y
_ ~ ' ~ f = I
/
/
8
/
°~°
4
l.ow Affinity System
/e 2
System
Knn"780-+120~M Vmax=68'5-+I=3.7 ,
Frog Spina/Cord
',v6
/- -e
Krn-760 + 120/zA,1 Vmox-77.6 -+7.8
~-If--i 0.2
i 0.4
i 016
i 018
i ] l0
i ] .2
I/[ToUrine] x 16 5 M
Fig. 4. Double reciprocal plots of [SH]taurine uptake in frog brain and frog spinal cord synaptosomes. Experimental conditions are as described in Methods. In the case of whole brain uptake the plot could be resolved into 2 straight-line components. The K,n and Vm=x values reported are means ± S.E.M. of 4 determinations. Velocity (V) is expressed as nmoles per nag protein/4 rain.
496 To evaluate the possibility that glycine accumulation might in fact be labeling 'taurinergic' elements, we examined for the presence of high affinity [3H]taurine uptake in frog spinal cord and brain (Fig. 4). No evidence for a high affinity accumulation of [3H]taurine can be demonstrated in frog spinal cord. Only a low affinity system is apparent with a Km value of 760 #M. This agrees with the failure of Davidoff and Adair to demonstrate high affinity [aH]taurine uptake by slices of frog spinal cord 11. In repeated experiments with homogenates of whole frog brain, a high affinity uptake system for taurine is apparent with a Km value of about 66 ,uM. This system can be distinguished from a low affinity uptake system with a Km value of 780 #M, similar to the Km of the low affinity uptake for taurine in frog spinal cord. DISCUSSION The present study revealed substantial amounts of high affinity glycine uptake by synaptosomal fractions and [3H]strychnine binding to synaptic membrane fractions in higher centers of frog brain as well as in frog spinal cord. This agrees with the observations of Davidoff and Adair10,1~ in slices of frog cerebral cortex and optic tectum showing the presence of high affinity accumulation of [aH]glycine and its potassium-evoked release. In the frog, the ratio for levels of glycine uptake and strychnine binding in whole brain to spinal cord is substantially higher in frog than in rat or chicken. Part of this difference can be attributed to the fact that the cerebral cortex, which is most deficient in 'glycinergic' systems, occupies a greater proportion of the whole brain in the rat than in the frog. However, an evaluation of discrete brain regions indicates that, even in the cerebral cortex, substantial levels of glycine uptake and strychnine binding are demonstrable in the frog but not in the rat. Thus, it is conceivable that in the frog glycine may play a synaptic role in higher centers as well as in the spinal cord and brain stem. Because of neurophysiological evidence suggesting that taurine rather than glycine may be the principal inhibitory transmitter in the frog spinal cord, we evaluated the properties of [3H]strychnine binding in the frog and rat. We found no difference in the substrate specificity of specific [all]strychnine binding in frog, chicken and rat spinal cord. Moreover, we were unable to detect high affinity accumulation of [aH]taurine by synaptosomal fractions of frog spinal cord, despite substantial levels of high affinity glycine uptake displayed by the same fractions. It is conceivable that taurine receptor binding sites and synaptosomal uptake sites exist but do not survive the procedures used in preparing brain tissue. Perhaps the greater potency of taurine than of glycine in inhibiting frog spinal motor neurons 24,26 may relate to the absence of an uptake system which would inactivate iontophoreticaUy-applied amino acids. Of the small neutral amino acids whose neurophysiological actions mimic those of glycine, L-a-alanine, L-proline, 7-aminobutyric acid and taurine have much less affinity than glycine for strychnine binding sites, fl-Alanine, on the other hand, has about the same affinity as glycine for the binding sites. However, endogenous levels of fl-alanine in mammalian CNS are extremely lowa°. We do not know of studies of endogenous fl-alanine levels in the frog spinal cord.
497 ACKNOWLEDGEMENTS Supported by U S P H S G r a n t MH-18501 a n d R S D A A w a r d MH-33128 to S.H.S. a n d a Deutsche Forschungsgemeinschaft fellowship to W . E . M .
REFERENCES 1 Aprison, M. N. and Werman, R., The distribution of glycine in cat spinal cord and roots, Life Sci., 4 (1965) 2075-2083. 2 Arregui, A., Logan, W. J., Bennett, J. P. and Sn~der, S. H., Specific glycine-accumulating synaptosomes in the spinal cord of rats, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 3485-3489. 3 Biscoe, T. J. and Curtis, D. R., Strychnine and cortical inhibition, Nature (Lond.), 214 (1967) 914-915. 4 Borg, J., Balcar, V. J. and Mandel, P., High affinity uptake of taurine in neuronal and glial cells, Brain Research, 118 (1976) 514-516. 5 Bradley, K., Easton, D. M. and Eccles, J. C., An investigation of primary or direct inhibition, J. Physiol. (Lond.), 122 (1953) 474-488. 6 Burt, D. R., Enna, S. J., Creese, J. and Snyder, S. H., Dopamine receptor binding in the corpus striatum of mammalian brain, Proc. nat. ,4cad. Sci. (Wash.), 72 (1972) 4655-4659. 7 Collins, G. G. S., The spontaneous and electrically-evoked release of [aH]GABA from isolated hemisected frog spinal cord, Brain Research, 66 (1974) 121-137. 8 Curtis, D. R., Hosli, L. and Johnston, G. A. R., A pharmacological study of the depression of spinal neurones by glycine and related amino acids, Exp. Brain Res., 6 (1968) 1-18. 9 Curtis, D. R., Hosli, L., Johnston, G. A. R. and Johnston, I. H., The hyperpolarization of spinal motoneurones by glycine and related amino acids, Exp. Brain Res., 5 (1968) 235-258. 10 Davidoff, R. A. and Adair, R., High affinity amino acid transport by frog spinal cord slices, J. Neurochem., 24 (1975) 545-552. I 1 Davidoff, R. A. and Adair, R., [SH]taurine accumulation by frog spinal cord slices, Brain Research, 110 (1976) 392-398. 12 Davidoff, R. A. and Adair, R., GABA and glycine transport in frog CNS: high affinity uptake and potassium-evoked release in vitro, Brain Research, 118 (1976) 403-415. 13 Enna, S. J. and Snyder, S. H., Properties of )'-aminobutyricacid (GABA) receptor binding in rat brain synaptic membrane fractions, Brain Research, 100 (1975) 81-97. 14 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain, J. Neurochem., 13 (1966) 655-669. 15 Graham, L. T., Jr., Shank, R. P., Werman, R. and Aprison, M. H., Distribution of some synaptic transmitter suspects in cat spinal cord: glutamic acid, aspartic acid, 7-aminobutyric acid, glycine and glutamine, J. Neurochem., 14 (1967) 465-472. 16 Henn, F. A. and Hamberger, A., Glial cell function: uptake of transmitter substances, Proc. nat. ,4cad. Sci. (Wash.), 68 (1971) 2682-2690. 17 Iversen, L. L. and Kelly, J. S., Uptake and metabolism of 7-aminobutyric acid by neurones and glial cells, Biochem. Pharmacol., 24 (1975) 933-938. 18 Johnston, G. A. R. and Iversen, L. L., Glycine uptake in rat central nervous system slices and homogenates: evidence for different uptake systems in spinal cord and cerebral cortex, J. Neurochem., 18 (1971) 1951-1961. 19 Kelly, J. S. and Krnjevi6, K., The action of glycine on cortical neurones, Exp. Brain Res., 9 (1969) 155-163. 20 Krnjevi6, K., Randic, M. and Straughan, D. W., Pharmacology of cortical inhibition, J. Physiol. (Lond.), 184 (1966) 78-105. 21 Logan, W. J. and Snyder, S. H., High affinity uptake systems for glycine, glutamic and aspartic acids in synaptosomes of rat central nervous tissues, Brain Research, 42 (1972) 413-431. 22 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 23 Mulder, A. H. and Snyder, S. H., Potassium-induced release of amino acids from cerebral cortex and spinal cord slices of the rat, Brain Research, 76 (1974) 297-308.
498 24 Nicoll, R. A., Padjen, A. and Barker, J. L., Analysis of amino acid responses on frog motoneurones, Neuropharmacology, 15 (1976) 45-53. 25 Schon, F. and Kelly, J. S., Selective uptake of [aH]fl-alanine by glia: association with the glial uptake system for GABA, Brain Research, 86 (1975) 243-257. 26 Sonnhof, U., Gratke, P., Krumnikl, J., Linder, M. and Schindler, L., Inhibitory postsynaptic actions of taurine, GABA, and other amino acids on motoneurones of the isolated frog spinal cord, Brain Research, 100 (1975) 327-341. 27 Tebecis, A.K.anddiMaria, A.,Strychnine-sensitiveinhibitioninthemedullar~ reticular formation: evidence for glycine as an inhibitory transmitter, Brain Research, 40 (1972) 373-383. 28 Werman, R., Davidoff, R. A. and Aprison, M. H., Inhibitory action of glycine on spinal neurons in the cat, J. Neurophysiol., 31 (1968) 81-95. 29 Yates, R. A. and Taberner, P. V., Glutamic acid, GABA and their metabolising enzymes in the frog central nervous system, Brain Research, 84 (1975) 399-407. 30 Yoshino, Y., DeFeudis, F. V. and Elliott, K. A. C., Omega-amino acids in rat brain, Canad. J. Biochem., 48 (1970) 147-148. 31 Young, A. B. and Snyder, S. H., Strychnine binding associated with glycine receptors of the central nervous system, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2832-2836. 32 Young, A. B. and Snyder, S. H., Strychnine binding in rat spinal cord membranes associated with synaptic glycine receptors: cooperativity of glycine interactions, Molec. Pharmacol., 10 (1974) 790-809.
487
GLYCINE HIGH AFFINITY UPTAKE AND STRYCHNINE BINDING ASSOCIATED WITH GLYCINE RECEPTORS IN THE FROG CENTRAL NERVOUS SYSTEM
WALTER E. MOLLER and SOLOMON H. SNYDER* Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Md. 21205 (U.S.A.)
(Accepted July 14th, 1977)
SUMMARY Accumulation of [aH]glycine into synaptosomal fractions occurs by high affinity systems in cerebral cortex, optic tectum, brain stem and spinal cord of the frog. Specific [all]strychnine binding which appears associated with postsynaptic glycine receptors is also demonstrable in these regions. By contrast, only very low levels of strychnine binding and high affinity glycine uptake occur in higher centers of the rat central nervous system. The relative potencies of small neutral amino acids in competing for [SH]strychnine binding are similar in frog brain and spinal cord. No evidence for a high affinity accumulation of [3H]taurine by synaptosomal fractions of frog spinal cord can be demonstrated. These observations favor glycine rather than taurine as an inhibitory transmitter in frog spinal cord. Moreover, these findings suggest that glycine may have a synaptic role in higher brain centers in the frog.
INTRODUCTION A variety of neurophysiological and neurochemical evidence indicates that glycine is a major inhibitory neurotransmitter in the spinal cord and brain stem of the mammalian central nervous system. Both natural postsynaptic inhibition and the effects of glycine in spinal cord and brain stem are blocked selectively by strychnineS, s, 9,27,2s. High affinity, sodium-dependent uptake systems for glycine into unique synaptosomal fractions have been demonstrated in mammalian spinal cord and brain stem 2,1s,21. Accumulated [aH]glycine is released selectively by depolarization of spinal cord and brain stem slices ~a. Endogenous levels of glycine are highest in spinal cord * To whom reprint requests should be addressed.
488 and brain stem1,15. Sites associated with glycine receptors can be labeled in mammalian spinal cord and brain stem by the binding of [3H]strychnine31,32. While neurochemical and neurophysiological studies indicate unique synaptic actions of glycine in mammalian spinal cord and brain stem, glycine appears to lack such properties in mammalian higher centers such as the cerebral cortex. Strychninesensitive hyperpolarization of neurons is not readily demonstrated in mammalian cerebral cortex3,19,2°. The high affinity glycine uptake system into unique synaptosomal fractions appears to be absent in mammalian cerebral cortex2,1s,21. Only negligible depolarization-induced release of accumulated [aH]glycine can be demonstrated from rat cerebral cortex slices23. Mammalian cerebral cortex also appears to lack detectable strychnine binding associated with glycine receptors 31. Recent evidence suggests that in the frog glycinergic elements may play a major role in higher centers. High affinity, sodium-dependent accumulation of glycine into cerebral cortex slices of the frog has been demonstrated1°. Potassium depolarization releases accumulated [aH]glycine from slices of frog cerebral cortex 1~. Neurophysiological evidence suggests that taurine rather than glycine may mediate strychnine-blocked postsynaptic inhibition in the frog spinal cord. Taurine is more potent than glycine in hyperpolarizing frog motor neurons ~4,26. Moreover, the inhibitory effects of taurine are more sensitive to blockade by strychnine than are those of glycine ~6. Endogenous levels of taurine in frog spinal cord are substantially higher than those of glycine 7. To clarify the possible synaptic role of glycine and other neutral amino acids such as taurine in the frog central nervous system, we have evaluated the accumulation of [aH]glycine by synaptosomal fractions as well as apparent receptor binding of [all]strychnine. METHODS
Animals Bullfrogs (Rana catesbiana, 6"-8" body length), chicken (3 weeks old), and male Sprague-Dawley rats were used. The frogs were obtained from Mogul-ED Corporation of Oshkosh, Wisconsin.
Membrane preparation The animals were decapitated and the brains and spinal cords rapidly removed. Frog and rat brains were dissected by the methods of Yates and Taberner 29 or Glowinski and Iversen14 respectively. Synaptic membranes were prepared as previously described for rat brain la and rat spinal cord 31,az. The final membrane pellet was frozen at --20 °C. Protein was measured by the method of Lowry et al.2L
[3H]strychnine binding The binding of [all]strychnine to synaptic membranes was determined by a modification6 of a previously described procedure al,32 in order to economize on tissue. The membrane pellet was homogenized in ice-cold 0.05 M sodium-potassium
489 phosphate buffer containing 200 mM NaC1 at pH 7.1 s2, using a Brinkmann Polytron PT-10. For the standard binding assay, 1-ml aliquots of tissue suspension (about 0.4 mg protein/ml) were added to test tubes containing Jail]strychnine (final concentration about 6 nM) and various amounts of non-radioactive strychnine or other compounds. After an incubation at 4 °C for 10 min a2, three 250-#1 aliquots of each incubation mixture were removed to plastic microfuge tubes (Beckman, 0.4 ml capacity) and centrifuged for 2.5 rain in a Beckman Microfuge B. The supernatant was removed by aspiration and each pellet rinsed superficially once with 250/A ice-cold incubation buffer. The bottom of each microfuge tube was cut off into a scintillation vial; the pellets were dissolved in 1 ml Protosol (New England Nuclear) and radioactivity was determined in 10 ml LSC (Yorktown Research) by liquid scintillation spectrometry at a counting efficiency of 43 ~. Specific [3H]strychnine binding was obtained by subtracting from the total bound radioactivity the amount not displaced by high concentrations of strychnine (0.1 mM) or glycine (1 mM). Values for specific binding of [all]strychnine were the same whether strychnine or glycine was used as displacer.
Uptake studies Uptake of amino acids by nuclei-free 1:20 sucrose (0.32 M) homogenates was assayed as previously described 21. Aliquots (0.2 ml) of the homogenates were incubated for 4 min at 37 °C together with 2.8 ml of Krebs-Ringer-Tris.HCl (pH 7.4 at 37 °C), containing various concentrations of the radioactive amino acids. Incubation mixtures were rapidly cooled and centrifuged at 28,000 × g for 10 min. The supernatant fluid was discarded and the pellets washed once with ice-cold 0.15 M NaC1 solution, resuspended in 10 ml ice-cold 0.15 M NaCI, and recentrifuged at 48,000 × g for 10 min. The final pellets were dissolved in 1 ml Protosol and the radioactivity was determined in 10 ml LSC by liquid scintillation spectrometry. Under these conditions [3H]glycine is accumulated primarily into synaptosomal fractions of rat 2,21 and frog spinal cord (W. Miiller, in preparation).
Materials [all]strychnine (radioactive purity 90~, specific activity 8.7 Ci/mmole) was labelled by catalytic tritium exchange as described previouslyal. [aH]glycine (11 Ci/mmole) and [3H]taurine (18 Ci/mmole) were obtained from New England Nuclear and Amersham/Searle respectively. All other substances were obtained from commercial suppliers. RESULTS
Saturation of [3H]glycine uptake and [all]strychnine binding [SH]glycine is accumulated by synaptosomal fractions of frog brain and frog spinal cord in a saturable fashion (Fig. 1). Both in brain and spinal cord discrete high and low affinity systems can be demonstrated as distinct components in repeated experiments. The Km values (25-30/~M) for the high affinity uptake system are similar in brain and spinal cord and resemble values for high affinity glycine uptake
490
I0 -
Fro~ Broi__n
HighAffinity System 0.8 Km =24 8_+[.4~M Vmax=455 4-025 / •
I/v o~ •
~,~ / -
/~..~/•__/
LOW Affinity System
/l~'e/Km" 02 ~" 4"~ i
i
IC •
389 -+I)~M Vmox=24,85:i:2.21
r
i
i
i
i
Frog Spinol Cord
HighAfhnlty System 08 Km=290_+07ffM Vmex=608+014 /
•
OA e~
•
LOW Affinity System
02 ~ " - /
Km=292±24/'~M
4
Vme*~)5.18 ±0.57
~
/ , 0,2
O'6
1 0.4
0 r8
' 1.0
' 12
I
",5 ~[OLYC,NE] .,0 M
Fig. 1. Double reciprocal plot of pH]glycine uptake in frog brain and frog spinal cord homogenates. Uptake of various concentrations of [aH]glycine was measured as described in Methods. In each case the plot could be resolved into 2 straight-line components in 4 repeated experiments. The Km and Vm a* values reported are means ~: S.E.M. of 4 determinations. Velocity (V) is expressed as m o l e s per mg protein/4 min.
0_
6
bJ
o
•
specific
/
~02
o nonspecific ,=,
4
a z o m hl z z -to >-
r-~
FROG SPINAL C
o
O
R
D
~
Total Binding Sites:2.4 pmole/rng prot
~o.-,,
~ 0,1 >-
• ~ -
(0 I
r-~ i
i
i
i
i
20 40 60 [3H]-STRYC HN, NE
i
~
0 ~M]
i
i
I00
nM
"."X,,.
2
i
KD:II
I
,\
2
[SH]-STRYCHNINE BOUND[nM]
L
3
Fig. 2. Saturation of specific [all]strychnine binding to frog spinal cord membranes, pH]strychnine binding was determined using the standard assay described in Methods with increasing concentrations of [all]strychnine. Non-sp~ific binding was obtained from experiments conducted in the presence of 0.1 m M strychnine or 1 m M glycine. The points are means of thr¢~ det~minations which varied less than 20%. The experiment was replicated 3 times. Left: linear plot. Right: Scatchard analysis of specific pH]strychnine binding.
491
o
4
:~' "6
3
• specific o nonspecific
E ,m z
FROG BRAIN 2
o
"r
F-
'
20
40
60
80
I00
[3H].STRYCHNINE [n~ ~ 005 Z 0 004 °
Z 003
Total Binding Sites, lO pmole/mg protein KD:I6nM
L.\
~oo2
\
I--
•
,~ oo,
\ • I
I 2
I 3
p.]-s-r..,c..,.E BouNo[ 4 Fig. 3. Saturation of specific [all]strychnine binding to frog whole brain membranes. Experimental conditions were the same as in Fig. 2. Upper part: linear plot. Lower part: Scatcbard analysis of specific [all]strychnine binding. The experiment was replicated 3 times.
previously reported in mammalian spinal cord synaptosomal fractions 21. The calculated maximal number of uptake sites for the high affinity system is slightly higher in the spinal cord than the brain stem. Both Km and Vmax values for low affinity accumulation of glycine are similar in frog brain and spinal cord. Saturable binding of [all]strychnine is demonstrable in crude synaptic membrane preparations of both frog spinal cord and whole frog brain (Figs. 2 and 3). At 6 nM [all]strychnine, the concentration used in routine binding studies, total [all]strychnine binding is about 3 and 2 times higher than the values for non-specific binding taken in the presence of 1 mM glycine or 0.1 mM non-radioactive strychnine in spinal cord and brain respectively. In both spinal cord and brain membranes, nonspecific binding values increase linearly with [all]strychnine concentration. By contrast, specific binding plateaus at about 20 nM [all]strychnine with half-maximal values at about 8-12 nM. Dissociation constants (KD) calculated for [all]strychnine binding in frog spinal cord and brain are 11 nM and 16 riM, respectively.
492 TABLE I Regional distribution of high affinity [aH Jgb,cine uptake and [ 3H ]strychnine binding in frog and rat C N S
High affinity [3H]glycine uptake was determined using 5 different [aH]glycine concentrations over the range of 8-30 uM. [3H]strychnine binding was determined by the assay procedure described in Methods, using a [all]strychnine concentration of 6 nM. Values reported are means ± S.E.M. of 4 (uptake) or 5 (Jail]strychnine binding) determinations. Glycine uptake values for rat cerebral cortex are in parentheses, because accumulation in this area does not appear to involve a high affinity system. High affinity [3H]glycine uptake
: 3Hjstrychnine binding
Km ~: (10 5 M)
V. . . . (nmole/rng protein~4")
(pmole/mg protein)
Frog Spinal cord Brain stem Optic tectum Cerebral cortex
2.90 4.13 4.05 3.60
± ± ± ±
0.07 0.14 0.37 0.32
6.08 3.88 3.00 3.07
± -i ±
0.14 0.25* 0.25* 0.20",**
0.80 0.33 0.21 0.10
~ i ± £
Rat Spinal cord Brain stem Midbrain Cerebral cortex
4.30 4.58 4.72 (43.30
± i ± ±
0.14 0.17 0.13 5.5)
5.31 ~ 2.91 ± 1.78 ± (9.03 ±
0.10 0.02* 0.08",** 1.38)
0.54 0.45 0.13 0.01
± 0.03 ± 0.02* ± 0.01",** j_: 0.003",**
0.03 0.01" 0.01",** 0.10",**
* When compared with the spinal cord, P < 0.001. ** When compared with the brain stem, P < 0.05.
Regional variations in [3H]glyeine uptake and [ 3Hjstrychnine binding The substantial levels of high affinity glycine uptake and of [3H]strychnine binding in whole frog brain contrasts with the very low levels of both in rat brain. Accordingly, we c o m p a r e d high affinity glycine uptake and [3H]strychnine binding in different regions of frog brain (Table I). Saturable accumulation of [ZH]glycine and [3H]strychnine binding are demonstrable in frog cerebrum, optic tectum, brain stem and spinal cord. The Km value for glycine accumulation is similar in the 4 areas. The maximal n u m b e r of glycine accumulating sites is highest in the spinal cord with values in the brain stem being a b o u t 64 ~ of spinal cord levels and densities in the optic rectum and cerebral cortex, cortex being about half o f spinal cord levels. Specific [ZH]strychnine binding is also highest in the spinal cord, about 2.4 times higher than the next highest levels which occur in the brain stem. Strychnine binding levels in the optic tectum and cerebral cortex are one-quarter and one-eighth, respectively, o f values in the spinal cord. These findings differ markedly from results in the rat cerebral cortex, where no high affinity uptake of glycine can be demonstrated (Table I) 18,21. Moreover, in the rat only negligible amounts o f [all]strychnine binding can be demonstrated in the cerebral cortex (Table I). In the rat, as in the frog ,the brain stem does possess substantial levels of high affinity glycine accumulation and [all]strychnine binding (Table I).
493 TABLE II Glycine uptake and [all]strychnine binding in brain and spinal cord of rat, chicken andfrog
[aH]glycine uptake was determined using 10 different [aH]glycineconcentrations over the range of 8-1000/zM. Values are means 4- S.E.M. of 4 determinations. [all]strychnine binding was determined by the standard procedure described in Methods, using 8 different [all]strychnine concentrations as shown in Figs. 2 and 3. The total number of binding sites and the Ko was determined by Scatchard analysis (Figs. 2 and 3). Values are means of three determinations which varied less than 20 %. [ZH]glycine uptake High affinity system Km (10-5 M)
Rat brain Chicken brain Frog brain Rat spinal cord Chicken spinal cord Frog spinal cord
Low affinity system
Vmax Km (Izmole/mg (10 4 M) protein/4")
{ZH]strychnine binding
Maximal KD I'max binding (nM) (l~m°le/mg (pmole/mg protein~4') protein)
3.40±0.13 1.934-0.28 7.564-1.4 19.14-3.6
High affinity glycine uptake
Vmnx
Maximal strychnine binding
0.3
9
6.4
3.7010.20 1.294-0.04 7.845:0.7 11.94-1.1 0.2 2.484-0.14 4.55±0.23 3.194-0.1 24.84-2.2 1.0
12 16
6.5 4.6
4.654-0.16 5.705:0.07 4.434-0.3 20.14-0.8 1.9
13
3.0
4.184-0.10 2.604-0.07 6.694-0.4 11.24-1.1
0.95
16
2.7
2.904-0.07 6.084-0.14 2.924-0.2 15.14-0.5
2.4
11
2.5
Comparison o f [3H]glycine uptake and [all]strychnine binding in chicken, rat and frog
Because of the differences in the properties of glycine uptake and receptor binding in frog and rat, we sought to evaluate a different animal class. Accordingly, in parallel experiments we compared properties of accumulation of [aH]glycine and binding of [all]strychnine in spinal cord and whole brain of chicken, frog and rat (Table II). Both high and low affinity systems for [SH]glycine uptake are apparent in whole brain and spinal cord of all three species. Km values for high affinity [aH]glycine uptake are fairly similar in all species examined and in both brain and spinal cord. Differences among the species are apparent, however, in the ratio of the Vmax values for glycine accumulation in spinal cord and brain. In the rat the maximal number of uptake sites in the spinal cord is triple values for whole rat brain. In the chicken the spinal cord Vmax is about double the value in brain, while, in the frog, Vmax values are only 1.3 times higher in spinal cord than brain. In contrast to the marked differences among the three species in high affinity glycine accumulation in spinal cord and brain, Vmax values for low affinity glycine uptake do not markedly differentiate the three species. For rat, chicken and frog, the amount of low affinity glycine accumulation is similar in spinal cord and brain. The KD values for [3H]strychnine binding in brain and spinal cord of the three species are quite similar (Table II). In all three species the number of strychnine binding sites is greater in spinal cord than in whole brain. However, there are species differences in the ratio of spinal cord to whole brain. In the rat, maximal [3H]-
494 strychnine binding is 6.3 times higher in spinal cord than brain and in the chicken the corresponding ratio is 4.7. By contrast, in the frog, the number of spinal cord binding sites in only twice the number of whole brain binding sites. Relative amounts of high affinity glycine uptake and [all]strychnine binding display similarities among the three species. Thus, while the absolute values for the Vmax of high affinity [aH]glycine and [3H]strychnine binding in whole brain of rat, chicken and frog may vary more than 2-3-fold, the ratio of glycine uptake and strychnine binding is quite similar for the three species. In the spinal cord the Vm ~x of the high affinity glycine uptake system and the maximal number of strychnine binding sites vary among species more than two-fold, while the ratio of these values is similar for the three species. Though whole brain levels of both high affinity glycine uptake and strychnine binding are less than for spinal cord in all three species, the difference between brain and spinal cord is greater for strychnine binding than for glycine uptake. The proportionately higher levels of high affinity glycine uptake in whole brain relative to spinal cord might conceivably reflect glycine uptake by constituents other than 'glycinergic' neurons. Several workers have demonstrated high affinity sodiumdependent accumulations of amino acids by glial cells in the central nervous system4,16,~7,25. Substrate specificity o f jail]strychnine binding in ]rog, rat and chicken
Because of neurophysiologic evidence that taurine is more potent than glycine in eliciting synaptic hyperpolarization in the frog spinal cord, we compared the potencies of glycine, taurine and fl-alanine in competing for [all]strychnine binding to spinal cord membranes of chicken, rat and frog (Table III). The affinities ofglycine are fairly similar in spinal cord membranes from the three species with IC50 values of 42-72 #M. In all three species, taurine is substantially less potent than glycine in competing for [all]strychnine binding with IC50 values 2.4-4.4 times greater than those of glycine, flAlanine has about the same affinity as glycine for the strychnine binding sites in all three species. The affinity of strychnine itself is similar in the three species. To ascertain whether strychnine labels the same sites in frog spinal cord and TABLE Ill Substrate specificity of [all]strychnine binding in rat, chicken and frog spinal cord
Inhibition of [SH]strychninebinding to synaptic membranes by amino acids and unlabeled strychnine was determined using the standard procedure described in Methods. IC50values, the concentration of displacer which reduces binding of laH]strychnineby 50 %, were calculated by log-probitanalysis, using 4-5 different concentrations of the displacer. Values are means i S.E.M. of 4 determinations.
Rat Chicken Frog
Glycine IC5o (~M)
Taurine IC5o (I~M)
fl-A lanine 1Cso (I~M)
Strychnine IC~o (nM)
41.7 -4- 6.0 72.3 ± 11.5 55.3 4- 15.1
185.0 4- 2.9 188.3 i 33.7 130.0 4- 8.9
61.3 -4- 7.0 96.7 :~ 24.6 46.7 -l- 6.7
11.3 q- 1.8 14.7 -4- 1.3 10.2 4- 1.4
495 TABLE IV
Substrate specificity of [aHjstrychnine binding in frog CNS IC5o values were determined as described in Table III. Values are means 4- S.E.M. of 4 determinations
Amino acid
Frog brain IC5o (ttM)
Glycine fl-Alanine Taurine L-a-Alanine L-Proline y-Aminobutyric acid L-Glutamic acid Strychnine
33 38 135 > 500 >>500 >>500 >>500 0.01
Frog spinal cord IC5o (#M)
4- 4 ± 4 -4- 17
4- 0.005
55 4- 15 47 :E 7 130 -4- 9 > 500 >>500 >>500 >>500 0.01 4- 0.001
brain, we compared the substrate specificity of [3H]strychnine binding in these two regions (Table IV). The IC50 value of strychnine itself is about the same in whole brain and spinal cord membranes. Of a series of amino acids evaluated, glycine and flalanine have the highest affinities, which is about the same in brain and spinal cord. In both regions the IC50 value for taurine is about 2.4-4.4 times greater than that for glycine. L-a-alanine, L-proline, 7-aminobutyric acid and L-glutamic acid have negligible affinity for strychnine binding sites in frog brain or spinal cord.
Frog Brein 86I/
High Affinity System
Km.66.3.+6~ " - - ~ / " Vmox =12.4-+0.7
2-
f I0
e~"
,/
V 4-
~'~
Low Affini)y
_ ~ ' ~ f = I
/
/
8
/
°~°
4
l.ow Affinity System
/e 2
System
Knn"780-+120~M Vmax=68'5-+I=3.7 ,
Frog Spina/Cord
',v6
/- -e
Krn-760 + 120/zA,1 Vmox-77.6 -+7.8
~-If--i 0.2
i 0.4
i 016
i 018
i ] l0
i ] .2
I/[ToUrine] x 16 5 M
Fig. 4. Double reciprocal plots of [SH]taurine uptake in frog brain and frog spinal cord synaptosomes. Experimental conditions are as described in Methods. In the case of whole brain uptake the plot could be resolved into 2 straight-line components. The K,n and Vm=x values reported are means ± S.E.M. of 4 determinations. Velocity (V) is expressed as nmoles per nag protein/4 rain.
496 To evaluate the possibility that glycine accumulation might in fact be labeling 'taurinergic' elements, we examined for the presence of high affinity [3H]taurine uptake in frog spinal cord and brain (Fig. 4). No evidence for a high affinity accumulation of [3H]taurine can be demonstrated in frog spinal cord. Only a low affinity system is apparent with a Km value of 760 #M. This agrees with the failure of Davidoff and Adair to demonstrate high affinity [aH]taurine uptake by slices of frog spinal cord 11. In repeated experiments with homogenates of whole frog brain, a high affinity uptake system for taurine is apparent with a Km value of about 66 ,uM. This system can be distinguished from a low affinity uptake system with a Km value of 780 #M, similar to the Km of the low affinity uptake for taurine in frog spinal cord. DISCUSSION The present study revealed substantial amounts of high affinity glycine uptake by synaptosomal fractions and [3H]strychnine binding to synaptic membrane fractions in higher centers of frog brain as well as in frog spinal cord. This agrees with the observations of Davidoff and Adair10,1~ in slices of frog cerebral cortex and optic tectum showing the presence of high affinity accumulation of [aH]glycine and its potassium-evoked release. In the frog, the ratio for levels of glycine uptake and strychnine binding in whole brain to spinal cord is substantially higher in frog than in rat or chicken. Part of this difference can be attributed to the fact that the cerebral cortex, which is most deficient in 'glycinergic' systems, occupies a greater proportion of the whole brain in the rat than in the frog. However, an evaluation of discrete brain regions indicates that, even in the cerebral cortex, substantial levels of glycine uptake and strychnine binding are demonstrable in the frog but not in the rat. Thus, it is conceivable that in the frog glycine may play a synaptic role in higher centers as well as in the spinal cord and brain stem. Because of neurophysiological evidence suggesting that taurine rather than glycine may be the principal inhibitory transmitter in the frog spinal cord, we evaluated the properties of [3H]strychnine binding in the frog and rat. We found no difference in the substrate specificity of specific [all]strychnine binding in frog, chicken and rat spinal cord. Moreover, we were unable to detect high affinity accumulation of [aH]taurine by synaptosomal fractions of frog spinal cord, despite substantial levels of high affinity glycine uptake displayed by the same fractions. It is conceivable that taurine receptor binding sites and synaptosomal uptake sites exist but do not survive the procedures used in preparing brain tissue. Perhaps the greater potency of taurine than of glycine in inhibiting frog spinal motor neurons 24,26 may relate to the absence of an uptake system which would inactivate iontophoreticaUy-applied amino acids. Of the small neutral amino acids whose neurophysiological actions mimic those of glycine, L-a-alanine, L-proline, 7-aminobutyric acid and taurine have much less affinity than glycine for strychnine binding sites, fl-Alanine, on the other hand, has about the same affinity as glycine for the binding sites. However, endogenous levels of fl-alanine in mammalian CNS are extremely lowa°. We do not know of studies of endogenous fl-alanine levels in the frog spinal cord.
497 ACKNOWLEDGEMENTS Supported by U S P H S G r a n t MH-18501 a n d R S D A A w a r d MH-33128 to S.H.S. a n d a Deutsche Forschungsgemeinschaft fellowship to W . E . M .
REFERENCES 1 Aprison, M. N. and Werman, R., The distribution of glycine in cat spinal cord and roots, Life Sci., 4 (1965) 2075-2083. 2 Arregui, A., Logan, W. J., Bennett, J. P. and Sn~der, S. H., Specific glycine-accumulating synaptosomes in the spinal cord of rats, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 3485-3489. 3 Biscoe, T. J. and Curtis, D. R., Strychnine and cortical inhibition, Nature (Lond.), 214 (1967) 914-915. 4 Borg, J., Balcar, V. J. and Mandel, P., High affinity uptake of taurine in neuronal and glial cells, Brain Research, 118 (1976) 514-516. 5 Bradley, K., Easton, D. M. and Eccles, J. C., An investigation of primary or direct inhibition, J. Physiol. (Lond.), 122 (1953) 474-488. 6 Burt, D. R., Enna, S. J., Creese, J. and Snyder, S. H., Dopamine receptor binding in the corpus striatum of mammalian brain, Proc. nat. ,4cad. Sci. (Wash.), 72 (1972) 4655-4659. 7 Collins, G. G. S., The spontaneous and electrically-evoked release of [aH]GABA from isolated hemisected frog spinal cord, Brain Research, 66 (1974) 121-137. 8 Curtis, D. R., Hosli, L. and Johnston, G. A. R., A pharmacological study of the depression of spinal neurones by glycine and related amino acids, Exp. Brain Res., 6 (1968) 1-18. 9 Curtis, D. R., Hosli, L., Johnston, G. A. R. and Johnston, I. H., The hyperpolarization of spinal motoneurones by glycine and related amino acids, Exp. Brain Res., 5 (1968) 235-258. 10 Davidoff, R. A. and Adair, R., High affinity amino acid transport by frog spinal cord slices, J. Neurochem., 24 (1975) 545-552. I 1 Davidoff, R. A. and Adair, R., [SH]taurine accumulation by frog spinal cord slices, Brain Research, 110 (1976) 392-398. 12 Davidoff, R. A. and Adair, R., GABA and glycine transport in frog CNS: high affinity uptake and potassium-evoked release in vitro, Brain Research, 118 (1976) 403-415. 13 Enna, S. J. and Snyder, S. H., Properties of )'-aminobutyricacid (GABA) receptor binding in rat brain synaptic membrane fractions, Brain Research, 100 (1975) 81-97. 14 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain, J. Neurochem., 13 (1966) 655-669. 15 Graham, L. T., Jr., Shank, R. P., Werman, R. and Aprison, M. H., Distribution of some synaptic transmitter suspects in cat spinal cord: glutamic acid, aspartic acid, 7-aminobutyric acid, glycine and glutamine, J. Neurochem., 14 (1967) 465-472. 16 Henn, F. A. and Hamberger, A., Glial cell function: uptake of transmitter substances, Proc. nat. ,4cad. Sci. (Wash.), 68 (1971) 2682-2690. 17 Iversen, L. L. and Kelly, J. S., Uptake and metabolism of 7-aminobutyric acid by neurones and glial cells, Biochem. Pharmacol., 24 (1975) 933-938. 18 Johnston, G. A. R. and Iversen, L. L., Glycine uptake in rat central nervous system slices and homogenates: evidence for different uptake systems in spinal cord and cerebral cortex, J. Neurochem., 18 (1971) 1951-1961. 19 Kelly, J. S. and Krnjevi6, K., The action of glycine on cortical neurones, Exp. Brain Res., 9 (1969) 155-163. 20 Krnjevi6, K., Randic, M. and Straughan, D. W., Pharmacology of cortical inhibition, J. Physiol. (Lond.), 184 (1966) 78-105. 21 Logan, W. J. and Snyder, S. H., High affinity uptake systems for glycine, glutamic and aspartic acids in synaptosomes of rat central nervous tissues, Brain Research, 42 (1972) 413-431. 22 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 23 Mulder, A. H. and Snyder, S. H., Potassium-induced release of amino acids from cerebral cortex and spinal cord slices of the rat, Brain Research, 76 (1974) 297-308.
498 24 Nicoll, R. A., Padjen, A. and Barker, J. L., Analysis of amino acid responses on frog motoneurones, Neuropharmacology, 15 (1976) 45-53. 25 Schon, F. and Kelly, J. S., Selective uptake of [aH]fl-alanine by glia: association with the glial uptake system for GABA, Brain Research, 86 (1975) 243-257. 26 Sonnhof, U., Gratke, P., Krumnikl, J., Linder, M. and Schindler, L., Inhibitory postsynaptic actions of taurine, GABA, and other amino acids on motoneurones of the isolated frog spinal cord, Brain Research, 100 (1975) 327-341. 27 Tebecis, A.K.anddiMaria, A.,Strychnine-sensitiveinhibitioninthemedullar~ reticular formation: evidence for glycine as an inhibitory transmitter, Brain Research, 40 (1972) 373-383. 28 Werman, R., Davidoff, R. A. and Aprison, M. H., Inhibitory action of glycine on spinal neurons in the cat, J. Neurophysiol., 31 (1968) 81-95. 29 Yates, R. A. and Taberner, P. V., Glutamic acid, GABA and their metabolising enzymes in the frog central nervous system, Brain Research, 84 (1975) 399-407. 30 Yoshino, Y., DeFeudis, F. V. and Elliott, K. A. C., Omega-amino acids in rat brain, Canad. J. Biochem., 48 (1970) 147-148. 31 Young, A. B. and Snyder, S. H., Strychnine binding associated with glycine receptors of the central nervous system, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2832-2836. 32 Young, A. B. and Snyder, S. H., Strychnine binding in rat spinal cord membranes associated with synaptic glycine receptors: cooperativity of glycine interactions, Molec. Pharmacol., 10 (1974) 790-809.
