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Substance P-evoked release of amino acid transmitters from the newborn rat spinal cord
354
Regulato 0 Pept~des, 46 (1993) 354-356 © 1993 Elsevier Science Pubhshers B V All rights reserved 0167-0115/93/$06 00
REGPEP 01459
Substance P-evoked release of amino acid transmitters from the newborn rat spinal cord T Maehara b, H. Suzuki a, K. Y o s h l o k a a a n d M. O t s u k a a Departments of ~Pharmacology and bNeurosurgery, Faculty of Medtcme, Tokyo Medzcal and Dental Umverstty, Tokyo (Japan)
Key words: GABA; Transmitter release; Transporter; Spinal cord
We have previously found that substance P (SP) evokes a release of G A B A from the newborn rat spinal cord and that this release has some atypical characteristics such as calcium-independence and tetrodotoxin-lnsensitivity Moreover, a G A B A uptake inhibitor, cis-4-hydroxympecot~c acid, which markedly augmented the G A B A release evoked by high K + , did not increase the SP-evoked G A B A release. Based on these findings we suggested that SP induces the G A B A release through a mechanism mvolving G A B A transporters [1] In this study we further examined the mechantsm of the SP-evoked G A B A release and also tested whether SP releases other amino acid transmitter candidates from the spinal cord Spinal cords below cervical segments were isolated from 1-4-day-old Wlstar rats under ether anesthesla. One hemisected cord was placed an a perfusion chamber, and continuously perfused w~th artificial cerebrospinal fluid (CSF). After washing the spinal cord for 2 h with the perfuslon medmm, 3-mln fractions were collected and amino acid contents
Correspondence to K Yoshloka, Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental Umverslty, 1-5-45 Yushlma, Bunkyo-ku, Tokyo 113, Japan
were determined using H P L C [1] The composition of artificial C S F was as follows, in m M ' NaC1 138.6, KC1 3.35, CaCI 2 1 26, MgC12 1.15, NaHCO3 21.0, NaH2PO 4 0.58, glucose 10.0. Bath application of SP (10 #M) as well as high K + (90 mM) medium evoked an increase m G A B A release from the spinal cord (Fig 1). Since G A B A transporters are known to be Na +- and C1-dependent [2,3], we examined the effects of low N a + medmm and low C1 - medium on the G A B A release Whde both the low N a ÷ and low C1- media markedly mcreased the G A B A release evoked by high K +, they depressed the SP-evoked G A B A release. The release of other amino acid transmitter candidates, glutamate, aspartate and glyclne, was also increased by SP to about 200°0 or more of the basal levels (Fig. 2). Furthermore, SP reduced a marked increase in the release of taurine and arginine, slight increase m the release of threonine but no changes m the release of alanine and tyroslne. The release of glutamate and glycine evoked by high K + was slgmficantly depressed in Ca 2 + -free medium (Ca 2÷ , 0 m M and Mg 2 +, 2.42 mM) and markedly augmented in the low N a + medium, whereas the SP-evoked release of these amino acids was neither depressed in the Ca 2+-free medium nor augmented in the low Na ÷ medmm (data not shown)
355
K+ - low Na÷
A
B
Na+ 22raM
11
K*-
It
I
20
I
"~" 20-
E
low CI"
CI 8mM
t
®g
~,o
Substance P -- low Na* ¢
I
2
E
b
Substance P - low CI
Na* 22mM
~
_~2,
t Cl" 8raM
]
E
!!1
E
Fig 1 The release of GABA evoked by high K + and SP from the newborn rat spinal cord and the effects of low Na ÷ medium and low CI - medmm High K ÷ (90 mM) medium or SP (10 mM) was apphed for 6 mm Each column and vertical bar represent the mean + S E M (n = 3-6)_ The basal values (open columns) are mean of two consecutive 3 mm-fractlons before apphcatlon of high K ÷ or SP and the values in the presence of the agent (filled columns) are mean of three 3 ram-fractions after apphcatlon of the agent After the first stimulation with high K + or SP the spinal cord was perfused wath normal artificial CSF for 30 mm and then perfused with the low Na ÷ or CI- medium for 30 mln before the second stimulus Immediately after the second stimulus normal artificial CSF was perfused for 1 h before the third stimulus The low Na ÷ (22 mM) medium was prepared by eqmmolar substitution of NaCI by chohne chloride and the low CI- (8 mM) medium was made by equlmolar substitution of NaC1 by Na methanesulfonate * P < 0 05 and **P<0_01 when compared with the precontrol release by high K + or SP
40-
30
2~ 0 Glu
Asp
GABA
GPy
Tau
Arg
Thr
Ala
Tyr
Fig 2 The S P - e v o k e d r c l c a s e o f ~ u t a m a t e ( G ] u ) , a s p a r t a t e ( A s p ) ,
GABA, glyclne (Oly), taurlne (Tau), argmme (Arg), threonine (Thr), alanme (Ala) and tyrosme (Tyr) * P < 0 05, **P<0.01 and ***P<0 001, when compared with the basal value_ The meaning of open and filled columns is the same as in Fig 1 (n = 13)
The reflux or efflux o f G A B A via G A B A transporters are k n o w n to d e p e n d on extracellular and intracellular concentrations o f N a ÷ , C1- and G A B A [2,3]. W h e n the extracellular N a ÷ or C1- concentration is reduced, this would result in a decrease in the G A B A influx, i.e., uptake, through G A B A transporters. This m a y provide an explanation for the increased overflow o f G A B A when the release is evoked by high K + in N a +- or C1--deficlent medium The reduction o f extracellular N a ÷ or C1concentration would also cause a reduction o f intracellular N a ÷ or C1- concentration since the influxes of these ions would be r e d u c e d under these conditions This m a y result in a reduction of efflux of
356 G A B A through transporters. This may explain why
References
the S P-evoked G A B A release was depressed m N a + or C1--deficient medium, provided that SP releases G A B A through transporters
By contrast, the high
K + -evoked G A B A ettlux is probably due to a C a 2 + de pe n d en t exocytosis and therefore would not be affected by a reduction of lntracellular N a + or C1concentration. Furthermore, the C a 2 + -independence and N a + - d e p e n d e n c e of the S P - e v o k e d glutamate and glycine release similar to those of the G A B A release suggest the existence o f a c o m m o n mechanism m the S P - e v o k e d release of amino acid transmitters In the n e w b o r n rat spinal cord.
1 Sakuma, M, Yoshloka, K, Suzuki, H, Yanagisawa, M, Onishl, Y, Kobayashl, N and Otsuka, M, Substance P-evoked release of GABA from isolated spinal cord of the newborn rat, Neurosclence, 45 (1991) 323-330 2 Radlan, R and Kanner, B I, Stolehlometry of sodmm- and chloride-coupled y-aminobutync acid transport by synaptlc plasma membrane vesicles isolated from rat brain, Biochemistry, 22 (1983) 1236-1241 3 Guastella, J, Nelson, N,, Nelson, H, Czyzyk, L, Keynan, S, Mledel, M C, Davxdson, N, Lester, H A and Kanner, B I, Cloning and expression of a rat brain GABA transporter, Science, 249 (1990) 1303-1306
Regulato 0 Pept~des, 46 (1993) 354-356 © 1993 Elsevier Science Pubhshers B V All rights reserved 0167-0115/93/$06 00
REGPEP 01459
Substance P-evoked release of amino acid transmitters from the newborn rat spinal cord T Maehara b, H. Suzuki a, K. Y o s h l o k a a a n d M. O t s u k a a Departments of ~Pharmacology and bNeurosurgery, Faculty of Medtcme, Tokyo Medzcal and Dental Umverstty, Tokyo (Japan)
Key words: GABA; Transmitter release; Transporter; Spinal cord
We have previously found that substance P (SP) evokes a release of G A B A from the newborn rat spinal cord and that this release has some atypical characteristics such as calcium-independence and tetrodotoxin-lnsensitivity Moreover, a G A B A uptake inhibitor, cis-4-hydroxympecot~c acid, which markedly augmented the G A B A release evoked by high K + , did not increase the SP-evoked G A B A release. Based on these findings we suggested that SP induces the G A B A release through a mechanism mvolving G A B A transporters [1] In this study we further examined the mechantsm of the SP-evoked G A B A release and also tested whether SP releases other amino acid transmitter candidates from the spinal cord Spinal cords below cervical segments were isolated from 1-4-day-old Wlstar rats under ether anesthesla. One hemisected cord was placed an a perfusion chamber, and continuously perfused w~th artificial cerebrospinal fluid (CSF). After washing the spinal cord for 2 h with the perfuslon medmm, 3-mln fractions were collected and amino acid contents
Correspondence to K Yoshloka, Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental Umverslty, 1-5-45 Yushlma, Bunkyo-ku, Tokyo 113, Japan
were determined using H P L C [1] The composition of artificial C S F was as follows, in m M ' NaC1 138.6, KC1 3.35, CaCI 2 1 26, MgC12 1.15, NaHCO3 21.0, NaH2PO 4 0.58, glucose 10.0. Bath application of SP (10 #M) as well as high K + (90 mM) medium evoked an increase m G A B A release from the spinal cord (Fig 1). Since G A B A transporters are known to be Na +- and C1-dependent [2,3], we examined the effects of low N a + medmm and low C1 - medium on the G A B A release Whde both the low N a ÷ and low C1- media markedly mcreased the G A B A release evoked by high K +, they depressed the SP-evoked G A B A release. The release of other amino acid transmitter candidates, glutamate, aspartate and glyclne, was also increased by SP to about 200°0 or more of the basal levels (Fig. 2). Furthermore, SP reduced a marked increase in the release of taurine and arginine, slight increase m the release of threonine but no changes m the release of alanine and tyroslne. The release of glutamate and glycine evoked by high K + was slgmficantly depressed in Ca 2 + -free medium (Ca 2÷ , 0 m M and Mg 2 +, 2.42 mM) and markedly augmented in the low N a + medium, whereas the SP-evoked release of these amino acids was neither depressed in the Ca 2+-free medium nor augmented in the low Na ÷ medmm (data not shown)
355
K+ - low Na÷
A
B
Na+ 22raM
11
K*-
It
I
20
I
"~" 20-
E
low CI"
CI 8mM
t
®g
~,o
Substance P -- low Na* ¢
I
2
E
b
Substance P - low CI
Na* 22mM
~
_~2,
t Cl" 8raM
]
E
!!1
E
Fig 1 The release of GABA evoked by high K + and SP from the newborn rat spinal cord and the effects of low Na ÷ medium and low CI - medmm High K ÷ (90 mM) medium or SP (10 mM) was apphed for 6 mm Each column and vertical bar represent the mean + S E M (n = 3-6)_ The basal values (open columns) are mean of two consecutive 3 mm-fractlons before apphcatlon of high K ÷ or SP and the values in the presence of the agent (filled columns) are mean of three 3 ram-fractions after apphcatlon of the agent After the first stimulation with high K + or SP the spinal cord was perfused wath normal artificial CSF for 30 mm and then perfused with the low Na ÷ or CI- medium for 30 mln before the second stimulus Immediately after the second stimulus normal artificial CSF was perfused for 1 h before the third stimulus The low Na ÷ (22 mM) medium was prepared by eqmmolar substitution of NaCI by chohne chloride and the low CI- (8 mM) medium was made by equlmolar substitution of NaC1 by Na methanesulfonate * P < 0 05 and **P<0_01 when compared with the precontrol release by high K + or SP
40-
30
2~ 0 Glu
Asp
GABA
GPy
Tau
Arg
Thr
Ala
Tyr
Fig 2 The S P - e v o k e d r c l c a s e o f ~ u t a m a t e ( G ] u ) , a s p a r t a t e ( A s p ) ,
GABA, glyclne (Oly), taurlne (Tau), argmme (Arg), threonine (Thr), alanme (Ala) and tyrosme (Tyr) * P < 0 05, **P<0.01 and ***P<0 001, when compared with the basal value_ The meaning of open and filled columns is the same as in Fig 1 (n = 13)
The reflux or efflux o f G A B A via G A B A transporters are k n o w n to d e p e n d on extracellular and intracellular concentrations o f N a ÷ , C1- and G A B A [2,3]. W h e n the extracellular N a ÷ or C1- concentration is reduced, this would result in a decrease in the G A B A influx, i.e., uptake, through G A B A transporters. This m a y provide an explanation for the increased overflow o f G A B A when the release is evoked by high K + in N a +- or C1--deficlent medium The reduction o f extracellular N a ÷ or C1concentration would also cause a reduction o f intracellular N a ÷ or C1- concentration since the influxes of these ions would be r e d u c e d under these conditions This m a y result in a reduction of efflux of
356 G A B A through transporters. This may explain why
References
the S P-evoked G A B A release was depressed m N a + or C1--deficient medium, provided that SP releases G A B A through transporters
By contrast, the high
K + -evoked G A B A ettlux is probably due to a C a 2 + de pe n d en t exocytosis and therefore would not be affected by a reduction of lntracellular N a + or C1concentration. Furthermore, the C a 2 + -independence and N a + - d e p e n d e n c e of the S P - e v o k e d glutamate and glycine release similar to those of the G A B A release suggest the existence o f a c o m m o n mechanism m the S P - e v o k e d release of amino acid transmitters In the n e w b o r n rat spinal cord.
1 Sakuma, M, Yoshloka, K, Suzuki, H, Yanagisawa, M, Onishl, Y, Kobayashl, N and Otsuka, M, Substance P-evoked release of GABA from isolated spinal cord of the newborn rat, Neurosclence, 45 (1991) 323-330 2 Radlan, R and Kanner, B I, Stolehlometry of sodmm- and chloride-coupled y-aminobutync acid transport by synaptlc plasma membrane vesicles isolated from rat brain, Biochemistry, 22 (1983) 1236-1241 3 Guastella, J, Nelson, N,, Nelson, H, Czyzyk, L, Keynan, S, Mledel, M C, Davxdson, N, Lester, H A and Kanner, B I, Cloning and expression of a rat brain GABA transporter, Science, 249 (1990) 1303-1306