N e ~ n c e Let~er~ 15 (I~79) 65--70 ©~;~4~ollaud Scient~e ~ L k h e m Ltd.
~.~OEF~~ CORD ~
E 8OMA~ATIN
65
RELF3~'E FROM RAT SPINAL
~
M. 8HE~AR~, ~ ~ Y
KRONHEIM,C.~)LINE ~ A M 8 and B. PI~tSTO~*
~ o ~ and i m m ~ Laboratory, D e ~ n t of ~edic~e, University of Cape 1 ' o ~ M ~ ~ o o l , &os~rvatory 7925 Cape To~,~. (;~vuth Africa)
Ac~pted ~1¥ . x ~ , 1979)
SUMMARY .~ ~ium-dependent releaseo~ immunoreactive ~omatostatin from rat spin~ cord i n ~ in ~ m m to two d e p o ~ stimuli(60 m M KCI and 75 ~ M vera~ne) has been d e m o ~ . Released so~atostatin immunoreactivitycomprim~ 0 . ~ of totaltissuecontent, showed parallelismwhen s~,,al dilutiom were c o m p m ~ to the immunoassay dose.response curve and el~,tmt gmn'lm~y to ~ynthetic somatostatin on Sephsdex G-25 (f) chromatog r a v y , These results ~ f ~ , h e r evidence for a neurotransmitter or neuzomodulator ml~ fc~ ~omato~tatin in m a r m u ~ ~ p ~ c o ~ . Soma~
has a widespread central nervous ay~em distribution [ 1] and
Immunonmoi~ty his been ~ m o ~ t n t ~ in ~ ~ cor~ by n,dioimmuno~ amy [9] ~ i m m u n ~ h ~ z i m y [5]. The p ~ . c e of the ~ e has been mown in human ~ with ~ cord or ~
~ ,
fluid [10,12] and in ~ of 24 patients c o ~ t m t i o n J of CSF immunozeactivity
were ~mve the ~ ~ level [12]. U~q~ the technique of ~ immunoflum~e~nce, s o ~ ~ immunonmc(~y hambeen found in some neumn~ cell bodies in spinal and in flbms in t ~ s ~ , ~
p~oftlm~cord
[5] ~td itlmsbeen
n e ~ m~kt~, a~d ~ ~mmtc~ta~m m a y play a role m an inhibitory t ~ ~ m ~ from p @ ~ ~ afferent nerve t e r m i n ~ in ml~-,It~m ~ coni [5]. Morn w c ~ t l y numemu, m ~ i n e ~ c ne~es have been shown to exist i n t h e grey matter of the spinal cord in addition to thorn demdbed in the s u ~ g e ~ of the columns po~znor,[4]. An impm~mt ¢¢Redon foz identificationof a t n z a m ~ is the demon. * ~
15thJauumT,19"/9
66 stration o~ release of the substance from nerve tenninak in response membrane d e p o t , on [11] and therefor~ the release of immunoreactive somato~ from rat spinal cord in vitro ~ bee~ studied. Unsexed Long.Evans rats ( ~ g ) , ~ u~ ~ t conditions and fed ad l=bitum, were ~ by decapitation between 09:00 ~ 1 0 : ~ h. The vertebral canals of the animals were exposed and s p ~ conis from thoracic and lumbar regions dissected free, removed arid i n d , : ~ ~ y preincubated in 5 m l ~ for 60 rain in 20 ml stoplz~Jd ~ ~ inan atmosphere of 9ff~ 02 ~ 5% CO2. The vials were ~laced in a ~ water bath at 37°C. Medium for incubation experiments comprised 1 ~ mM NaCI, 6 mM KCI, I mM Na2HPO4, 0.88 mbl MgSO4.7H20, 22 mM NaHCO3, 1.45 rnM CaC12, 11 mM D~lucose and 0.1% bovine serum albumin ( c r y ~ ; BDH). After preincubation spinal cords were t r a n s f e ~ to similarly stoppered glass vials containing 2 nd normal medium (as described) or 2 ml test medhun (i.e. with test s u ~ c e added). After 30 rain incubation at 37°C in a shaking water bath, the medium was decanted, boiled, map frozen end stored at --200C; somatostatin was meastm~ by radioimmunoassay as p t ~ o u s l y described [9]. A number of spinal cords were weighed following d i u e ~ o n and immunoreactive somatostatin content determiv~i followh~g extraction in 2 M acetic a~d [9]. Viability of the spinal cords was ~ by memmring oxygen ~ m p t i o n by the W ~ technique. Na~IPO4 (22 raM) was substituted for NaHCO3 in the incubation ~ and experim,mts were performed in an atmosphere of 100% O~. To assess enzymic degradation of releued immunoreactive s o ~ , the recovery of 10 rig ~mthetic somatostatln (Ayemt AY24910) ~ to the L~cub~tion medium was measur~ ~ 30 rain i n c u b l t ~ . PJq)er chromatoelectrophoresis [9] was performed on 1 2 ' I . T y ~ . ~ o ~ t i n incubated for 20 h (t_i~ of immunoassay incubation) at 4°C with assay buffer alone or medium removed after 3 0 ~ ~ubation ~ spi,,~ cot~ to assess de. Bradation of label by ~ e incubation medium. ~ t ~ e x ~ _ _~ts were performed ~ r the 60-rain preincubation p e ~ , To study ~ . r e ~ of somatosta_tin in ~ pre~nce of depolzdsing stimuli, ~ substances ~ r e made up in ~ ~ b s ~ i o n m ~ to give final concentrations of 6 0 mM K ~ 75/~M ~ ~ (Sigma). ~ ~ r experiments to determine the on ~ m ions, medium was made up to contah~ 60 mM KCI ~ K * ) ~ initio; in this high K* medium 20 #M verapsmfl hydrochloride (Isoptin, Knoll) was prepared or a Ca2*-free ~ i u m ~ obtained ~ on~tting CaCI=. To ~ ~ identity of re~e~sed immunoreactive s o m ~ i n with syn. the~ic somatostatin, ~erial dilutions of medium were compared to the ira. m u n o ~ y dose-~esponse curve for parallelism. ~.~ addition elution profiles were compared after S~phadex G-25 (f) chromatography in 1 M acetic acid ~n ~ ~ X ~ .6 cm colunm.
67
Because o f biological variation between exFeriments release of immunoreactive somatostatin in response to test s u ~ c e s was compared to release in contzol ~ in simultaneously p e d ~ o x ~ d experiments. Significant differences between test and contzol ~ x~sults were evaluated by Wil. coxon ~ test for unpaired n o n - ~ e t n c data (Mann-Whitney). Mean wet weight o f removed spinal cords was 0.14 -+ 0.01 g (mean + S.E.M0) and immunomactive s o m a t o ~ c o n t e n t 701.1 -+ 54.2 ng/g wet weight tissue (mean ± S.E.M., n = 15). Viability c f the tissue was sugges~ed by ~ oxygen consumption over 120 rain o f Lucubation (r = 0.9698, P < 0.001) with mean oxygen consumption o f 106.~', -+ 21.6/~mol O2/g • h -1 ~mean ± S . E . M . n = 4 ) . Of 10 ng s y n ~ e t i c s o - - t i n added to the medium 86.1 +- 15.4% (mean ± S.E_M., n = 4) was ~ c o v e ~ after 30 rain incubation in the presence of spinal cord. Incubation o f f ~ s h l y iodinated tracer (6.4 -+ 0.5% damage on paper chromatoelec~opho~msis) with medium from spin~i cord for the g o m a t o ~ i n immtmoamay incubation time (20 h) result~l in no significant excess in degradstion (11.2 ± 1.0% damage) compared to that seen in assay 0uffer alone (10.4 ± 1.4% dsmage; mean ± S.E.M., n = 4). The mean b ~ relea~ o f immunoreactive somatostatin was 0.52 ng/spinal cozd/30 rain and comprised 0.53% of total tissue content. In the presence of two d e p o ~ s~imuli (60 mM KCI and 75/~M veratfine) the release of immunoreactive s o m a ~ t i n w ~ significantly increased (Table I). The K*stimu~ mleame was ~ b i t e d , however, in the absence of CaCI2 from the medium and i~ the pv~ence of 20 ~M verapamil (an agent which interfere~ with calcium flux) in the medium (Table II). Serial dilutions ~'f released immunoreactivity ~howed parallelism to the dose.response ~ e (data not shown), S~phedex G.25 (f) chromatography revealed close identity between the majority of released immunoreactive somatostatin and synthetic somatotatin
TABLE I SOMATOSTATII~ RELEASE FROM RAT SPINAL CORD
Effect of depolari~J~g =timuli (KCi and veratrine) on immunoreactive somatostatin release from rat spinal cord in vitro. Remit= are expremmd ~ mean ± S.E~M.,number of experiment= in pazenthesiJ. Significant differences between test ~=d control release reJult~ are re~t~d by *P < 0.01. Somatogatin (ng/~aal eotdl30 rain) C~nt~rol KC160 mM Control Veratfine 75 ~M
0.52 • 1.08 0.64 • 1.32
± 0.04 ~ 0.12 ~ 0.10 ~ 0.14
(18) (16) (12) (15)
68
TABt~ H SOMATOSTATIN R ~ FROM RAT ~ N A L CORD Effect ~ ~ ofeale~. ~zioas from ~ ami ~ ~ f ~ 20 ~Moa pota~mm ( ~ 60 ~ ~ c i ~ i m m u n o m a z t i v e ~ reles~ frmn r ~ spinal cord in vitro. ~ m ~ e ~ ~ ~ • S.B.M., ~ ofe~tsm pare~~. 8igniflemzt diffe~mcm b e t ~ m test and c z m l a ~ ! ~ reults sre rept,e~mted by *P < 0.05 an& **P < 0.01. .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.,
i,
-
KC160 mM KC160 zhM ÷ C~ ~°-fr~ medium KCI t~0 ~LM
KCI e0 ~
.
1.04 • 0.56 0.84 • "0.41
÷ vmqmmfl 20 ~M
,
,
,
.
,
.
• 0.16 ( 9 ) z O.OS ( 9 ) • 0.12 (10) ± 0.06 ( 8 )
A n ~ . ~ r of centnd nervous ~stem peptides have been d e m o ~ b y mdloimmunommay fred i
m
m
u
n
~
t o be present in mare-
spinal conl; ~ ~ sometoststin, thym~pbin rele~in~ hormone [6], ~ c e P [7] ~ [8] and vuosct~e infzatJnal peptlde [2]. In sddltion the ~ c e o f soamomtin, vmoactive intestinsl
is] m i
md cho
kinin immuz z
,
[lS] in
human cerebrospinal fluid ( C S ¥ ) ~ been repoeted. ~ p h ~ l o g i c a l role of these pepttdemin spinal cord and CSF b n ~ defined M yet but with to substance P and s
o
~
it 1 ~ been ~ l l p d ~ d t h ~ ~
two peptJdu
0.3Vo 0.2"
~0-1'
150 100 ELUTI~ VOLIME (ML)
Fig. 1. ]bution protrde of ~ m u n o ~ t i v e somatostat~ released from spinal cord on Sepb.m:lez G-26 (f) ~ m a t o g r a p h y (I"o = void volume; 94 x 1.6 cm cohunn, flow rate 30 ml/h, fraction volume 5.0 ml).
69
m a y bare respective excitatory and inhibitory transmitter functions [5]. The calcium-dependent release of immunoreactive substance P f~om isolated rat spinal cord m response to stimulation of dorsal nerve roots h~s provided support for the view that substance P is a ph~ological transmitter released from dorsal root fibres [11], and our demonstration of a calcium~lependent depolarising stimulus-evoked release of immunoreactive somak)statm from incubated rat spinal cord provides further e~dence for a transmitter role for 3omatostatin in the spinal cord. ACKNOWLEDGEMENTS We t h a n k Mr. A. Isaacs a n d Mr. D. Petersen for their skillful assistance with animola. Financial s u p p o r t was o b t a i n e d from the S o u t h African Medical Research Council a nd A t o m i c Energy Board, In tern atio n al A to mic Energy A g e n c y C o n t r a c t No. 1 8 0 6 / R B and th e University of Cape T o w n Staff Research F u n d . We are grateful t o Professor R. Kitsch for constructive criticism during the p r e p a r a t i o n of the manuscript. REFERENCES
1 Brownstein, M., Arimura, A., S~to, H., Scbally, A.V. o~ld Kizer, J.S., The regional distribution of somatostatin ~ the rat brain, Endocrinology, 96 (1975) 1456--1461. 2 Fahrenkrug, J. and de Muckadell, O.B.S., Distribution of vasoactive intestinal polypeptide (VIP) in the porcine central nervous system, J. Neurochem., 31 (1978) 1445--1451. 3 Fahrenkrug, J., de Muckadell, O.B.S. and Fahrenkrug, A., Vasoactive intestinal polypeptide (VIP)in human cerebrospinal fluid, Brain Res., 124 (1977) 581--584. 4 Fora~nann, W.G., A new ~mato,tatinergic sy~d~emin the mammalian spinal cord, ' lrosci. Lett., 10 (1978) 293--297. 5 H0kfeit, T., Elde, R.~ J o h a n ~ n , O., Luft, R. and Arimura, A., Immunobistochemical evidence for the presence of somatostatin, a powerful inhibitory peptide, in some primary sensory neurons, Neurosci. Lett., I (1975) 231--235. 6 H6kfelt, T., Fuxe, Kp, Jonan~on, O., Jeffcoate, S. and White, N., Distribution of thyr~tropin-releuing hormone (TRH) in the central nervous system as revealed with i m m u n o h i s t o c h e m ~ , Europ. J. Pharmacol., 34 (1975) 389--392. 7 H6kfelt, T., Kelle~th, J.O., N ~ n , G. and Pernow, B., Substance P: localization in the central nervous ¢ y ~ m and in some primary sensory neurons, Science, 190 ( 1975) 889--890. 8 Kobaya~i, RAI., Brown, M. and Vale, W., Regional distribution of neurotensin a~d somatbstath in rat brain, Brain Res., 126 (1977) 584~588. 9 Kronheim, 8 , Berelowitz, M. and ~mstone, B.L., A r a d i o i m m u ~ y for growth hormone releme-inhibiting hormone: method and quantitative tissue distribution, Clin. Endocr., 5 (1976) 619--630. 10 Kronheim, S., Berelowitz, M. and Firestone, B.L., The presence of immunoreactive growth hormone relea~-inhibit~ h~mone in normal cerebrospinal fluid, Clin. Endocr., 6 (1977) 411-415.
11 Otsuka,M. ~ Konishi,S.~ Releaseof sul~tance Polikeimmunoreactivity from isolatedspinalcord of n~;wborn rat,Nature (Lond.),264 (1976) 83--84.
~.~OEF~~ CORD ~
E 8OMA~ATIN
65
RELF3~'E FROM RAT SPINAL
~
M. 8HE~AR~, ~ ~ Y
KRONHEIM,C.~)LINE ~ A M 8 and B. PI~tSTO~*
~ o ~ and i m m ~ Laboratory, D e ~ n t of ~edic~e, University of Cape 1 ' o ~ M ~ ~ o o l , &os~rvatory 7925 Cape To~,~. (;~vuth Africa)
Ac~pted ~1¥ . x ~ , 1979)
SUMMARY .~ ~ium-dependent releaseo~ immunoreactive ~omatostatin from rat spin~ cord i n ~ in ~ m m to two d e p o ~ stimuli(60 m M KCI and 75 ~ M vera~ne) has been d e m o ~ . Released so~atostatin immunoreactivitycomprim~ 0 . ~ of totaltissuecontent, showed parallelismwhen s~,,al dilutiom were c o m p m ~ to the immunoassay dose.response curve and el~,tmt gmn'lm~y to ~ynthetic somatostatin on Sephsdex G-25 (f) chromatog r a v y , These results ~ f ~ , h e r evidence for a neurotransmitter or neuzomodulator ml~ fc~ ~omato~tatin in m a r m u ~ ~ p ~ c o ~ . Soma~
has a widespread central nervous ay~em distribution [ 1] and
Immunonmoi~ty his been ~ m o ~ t n t ~ in ~ ~ cor~ by n,dioimmuno~ amy [9] ~ i m m u n ~ h ~ z i m y [5]. The p ~ . c e of the ~ e has been mown in human ~ with ~ cord or ~
~ ,
fluid [10,12] and in ~ of 24 patients c o ~ t m t i o n J of CSF immunozeactivity
were ~mve the ~ ~ level [12]. U~q~ the technique of ~ immunoflum~e~nce, s o ~ ~ immunonmc(~y hambeen found in some neumn~ cell bodies in spinal and in flbms in t ~ s ~ , ~
p~oftlm~cord
[5] ~td itlmsbeen
n e ~ m~kt~, a~d ~ ~mmtc~ta~m m a y play a role m an inhibitory t ~ ~ m ~ from p @ ~ ~ afferent nerve t e r m i n ~ in ml~-,It~m ~ coni [5]. Morn w c ~ t l y numemu, m ~ i n e ~ c ne~es have been shown to exist i n t h e grey matter of the spinal cord in addition to thorn demdbed in the s u ~ g e ~ of the columns po~znor,[4]. An impm~mt ¢¢Redon foz identificationof a t n z a m ~ is the demon. * ~
15thJauumT,19"/9
66 stration o~ release of the substance from nerve tenninak in response membrane d e p o t , on [11] and therefor~ the release of immunoreactive somato~ from rat spinal cord in vitro ~ bee~ studied. Unsexed Long.Evans rats ( ~ g ) , ~ u~ ~ t conditions and fed ad l=bitum, were ~ by decapitation between 09:00 ~ 1 0 : ~ h. The vertebral canals of the animals were exposed and s p ~ conis from thoracic and lumbar regions dissected free, removed arid i n d , : ~ ~ y preincubated in 5 m l ~ for 60 rain in 20 ml stoplz~Jd ~ ~ inan atmosphere of 9ff~ 02 ~ 5% CO2. The vials were ~laced in a ~ water bath at 37°C. Medium for incubation experiments comprised 1 ~ mM NaCI, 6 mM KCI, I mM Na2HPO4, 0.88 mbl MgSO4.7H20, 22 mM NaHCO3, 1.45 rnM CaC12, 11 mM D~lucose and 0.1% bovine serum albumin ( c r y ~ ; BDH). After preincubation spinal cords were t r a n s f e ~ to similarly stoppered glass vials containing 2 nd normal medium (as described) or 2 ml test medhun (i.e. with test s u ~ c e added). After 30 rain incubation at 37°C in a shaking water bath, the medium was decanted, boiled, map frozen end stored at --200C; somatostatin was meastm~ by radioimmunoassay as p t ~ o u s l y described [9]. A number of spinal cords were weighed following d i u e ~ o n and immunoreactive somatostatin content determiv~i followh~g extraction in 2 M acetic a~d [9]. Viability of the spinal cords was ~ by memmring oxygen ~ m p t i o n by the W ~ technique. Na~IPO4 (22 raM) was substituted for NaHCO3 in the incubation ~ and experim,mts were performed in an atmosphere of 100% O~. To assess enzymic degradation of releued immunoreactive s o ~ , the recovery of 10 rig ~mthetic somatostatln (Ayemt AY24910) ~ to the L~cub~tion medium was measur~ ~ 30 rain i n c u b l t ~ . PJq)er chromatoelectrophoresis [9] was performed on 1 2 ' I . T y ~ . ~ o ~ t i n incubated for 20 h (t_i~ of immunoassay incubation) at 4°C with assay buffer alone or medium removed after 3 0 ~ ~ubation ~ spi,,~ cot~ to assess de. Bradation of label by ~ e incubation medium. ~ t ~ e x ~ _ _~ts were performed ~ r the 60-rain preincubation p e ~ , To study ~ . r e ~ of somatosta_tin in ~ pre~nce of depolzdsing stimuli, ~ substances ~ r e made up in ~ ~ b s ~ i o n m ~ to give final concentrations of 6 0 mM K ~ 75/~M ~ ~ (Sigma). ~ ~ r experiments to determine the on ~ m ions, medium was made up to contah~ 60 mM KCI ~ K * ) ~ initio; in this high K* medium 20 #M verapsmfl hydrochloride (Isoptin, Knoll) was prepared or a Ca2*-free ~ i u m ~ obtained ~ on~tting CaCI=. To ~ ~ identity of re~e~sed immunoreactive s o m ~ i n with syn. the~ic somatostatin, ~erial dilutions of medium were compared to the ira. m u n o ~ y dose-~esponse curve for parallelism. ~.~ addition elution profiles were compared after S~phadex G-25 (f) chromatography in 1 M acetic acid ~n ~ ~ X ~ .6 cm colunm.
67
Because o f biological variation between exFeriments release of immunoreactive somatostatin in response to test s u ~ c e s was compared to release in contzol ~ in simultaneously p e d ~ o x ~ d experiments. Significant differences between test and contzol ~ x~sults were evaluated by Wil. coxon ~ test for unpaired n o n - ~ e t n c data (Mann-Whitney). Mean wet weight o f removed spinal cords was 0.14 -+ 0.01 g (mean + S.E.M0) and immunomactive s o m a t o ~ c o n t e n t 701.1 -+ 54.2 ng/g wet weight tissue (mean ± S.E.M., n = 15). Viability c f the tissue was sugges~ed by ~ oxygen consumption over 120 rain o f Lucubation (r = 0.9698, P < 0.001) with mean oxygen consumption o f 106.~', -+ 21.6/~mol O2/g • h -1 ~mean ± S . E . M . n = 4 ) . Of 10 ng s y n ~ e t i c s o - - t i n added to the medium 86.1 +- 15.4% (mean ± S.E_M., n = 4) was ~ c o v e ~ after 30 rain incubation in the presence of spinal cord. Incubation o f f ~ s h l y iodinated tracer (6.4 -+ 0.5% damage on paper chromatoelec~opho~msis) with medium from spin~i cord for the g o m a t o ~ i n immtmoamay incubation time (20 h) result~l in no significant excess in degradstion (11.2 ± 1.0% damage) compared to that seen in assay 0uffer alone (10.4 ± 1.4% dsmage; mean ± S.E.M., n = 4). The mean b ~ relea~ o f immunoreactive somatostatin was 0.52 ng/spinal cozd/30 rain and comprised 0.53% of total tissue content. In the presence of two d e p o ~ s~imuli (60 mM KCI and 75/~M veratfine) the release of immunoreactive s o m a ~ t i n w ~ significantly increased (Table I). The K*stimu~ mleame was ~ b i t e d , however, in the absence of CaCI2 from the medium and i~ the pv~ence of 20 ~M verapamil (an agent which interfere~ with calcium flux) in the medium (Table II). Serial dilutions ~'f released immunoreactivity ~howed parallelism to the dose.response ~ e (data not shown), S~phedex G.25 (f) chromatography revealed close identity between the majority of released immunoreactive somatostatin and synthetic somatotatin
TABLE I SOMATOSTATII~ RELEASE FROM RAT SPINAL CORD
Effect of depolari~J~g =timuli (KCi and veratrine) on immunoreactive somatostatin release from rat spinal cord in vitro. Remit= are expremmd ~ mean ± S.E~M.,number of experiment= in pazenthesiJ. Significant differences between test ~=d control release reJult~ are re~t~d by *P < 0.01. Somatogatin (ng/~aal eotdl30 rain) C~nt~rol KC160 mM Control Veratfine 75 ~M
0.52 • 1.08 0.64 • 1.32
± 0.04 ~ 0.12 ~ 0.10 ~ 0.14
(18) (16) (12) (15)
68
TABt~ H SOMATOSTATIN R ~ FROM RAT ~ N A L CORD Effect ~ ~ ofeale~. ~zioas from ~ ami ~ ~ f ~ 20 ~Moa pota~mm ( ~ 60 ~ ~ c i ~ i m m u n o m a z t i v e ~ reles~ frmn r ~ spinal cord in vitro. ~ m ~ e ~ ~ ~ • S.B.M., ~ ofe~tsm pare~~. 8igniflemzt diffe~mcm b e t ~ m test and c z m l a ~ ! ~ reults sre rept,e~mted by *P < 0.05 an& **P < 0.01. .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.,
i,
-
KC160 mM KC160 zhM ÷ C~ ~°-fr~ medium KCI t~0 ~LM
KCI e0 ~
.
1.04 • 0.56 0.84 • "0.41
÷ vmqmmfl 20 ~M
,
,
,
.
,
.
• 0.16 ( 9 ) z O.OS ( 9 ) • 0.12 (10) ± 0.06 ( 8 )
A n ~ . ~ r of centnd nervous ~stem peptides have been d e m o ~ b y mdloimmunommay fred i
m
m
u
n
~
t o be present in mare-
spinal conl; ~ ~ sometoststin, thym~pbin rele~in~ hormone [6], ~ c e P [7] ~ [8] and vuosct~e infzatJnal peptlde [2]. In sddltion the ~ c e o f soamomtin, vmoactive intestinsl
is] m i
md cho
kinin immuz z
,
[lS] in
human cerebrospinal fluid ( C S ¥ ) ~ been repoeted. ~ p h ~ l o g i c a l role of these pepttdemin spinal cord and CSF b n ~ defined M yet but with to substance P and s
o
~
it 1 ~ been ~ l l p d ~ d t h ~ ~
two peptJdu
0.3Vo 0.2"
~0-1'
150 100 ELUTI~ VOLIME (ML)
Fig. 1. ]bution protrde of ~ m u n o ~ t i v e somatostat~ released from spinal cord on Sepb.m:lez G-26 (f) ~ m a t o g r a p h y (I"o = void volume; 94 x 1.6 cm cohunn, flow rate 30 ml/h, fraction volume 5.0 ml).
69
m a y bare respective excitatory and inhibitory transmitter functions [5]. The calcium-dependent release of immunoreactive substance P f~om isolated rat spinal cord m response to stimulation of dorsal nerve roots h~s provided support for the view that substance P is a ph~ological transmitter released from dorsal root fibres [11], and our demonstration of a calcium~lependent depolarising stimulus-evoked release of immunoreactive somak)statm from incubated rat spinal cord provides further e~dence for a transmitter role for 3omatostatin in the spinal cord. ACKNOWLEDGEMENTS We t h a n k Mr. A. Isaacs a n d Mr. D. Petersen for their skillful assistance with animola. Financial s u p p o r t was o b t a i n e d from the S o u t h African Medical Research Council a nd A t o m i c Energy Board, In tern atio n al A to mic Energy A g e n c y C o n t r a c t No. 1 8 0 6 / R B and th e University of Cape T o w n Staff Research F u n d . We are grateful t o Professor R. Kitsch for constructive criticism during the p r e p a r a t i o n of the manuscript. REFERENCES
1 Brownstein, M., Arimura, A., S~to, H., Scbally, A.V. o~ld Kizer, J.S., The regional distribution of somatostatin ~ the rat brain, Endocrinology, 96 (1975) 1456--1461. 2 Fahrenkrug, J. and de Muckadell, O.B.S., Distribution of vasoactive intestinal polypeptide (VIP) in the porcine central nervous system, J. Neurochem., 31 (1978) 1445--1451. 3 Fahrenkrug, J., de Muckadell, O.B.S. and Fahrenkrug, A., Vasoactive intestinal polypeptide (VIP)in human cerebrospinal fluid, Brain Res., 124 (1977) 581--584. 4 Fora~nann, W.G., A new ~mato,tatinergic sy~d~emin the mammalian spinal cord, ' lrosci. Lett., 10 (1978) 293--297. 5 H0kfeit, T., Elde, R.~ J o h a n ~ n , O., Luft, R. and Arimura, A., Immunobistochemical evidence for the presence of somatostatin, a powerful inhibitory peptide, in some primary sensory neurons, Neurosci. Lett., I (1975) 231--235. 6 H6kfelt, T., Fuxe, Kp, Jonan~on, O., Jeffcoate, S. and White, N., Distribution of thyr~tropin-releuing hormone (TRH) in the central nervous system as revealed with i m m u n o h i s t o c h e m ~ , Europ. J. Pharmacol., 34 (1975) 389--392. 7 H6kfelt, T., Kelle~th, J.O., N ~ n , G. and Pernow, B., Substance P: localization in the central nervous ¢ y ~ m and in some primary sensory neurons, Science, 190 ( 1975) 889--890. 8 Kobaya~i, RAI., Brown, M. and Vale, W., Regional distribution of neurotensin a~d somatbstath in rat brain, Brain Res., 126 (1977) 584~588. 9 Kronheim, 8 , Berelowitz, M. and ~mstone, B.L., A r a d i o i m m u ~ y for growth hormone releme-inhibiting hormone: method and quantitative tissue distribution, Clin. Endocr., 5 (1976) 619--630. 10 Kronheim, S., Berelowitz, M. and Firestone, B.L., The presence of immunoreactive growth hormone relea~-inhibit~ h~mone in normal cerebrospinal fluid, Clin. Endocr., 6 (1977) 411-415.
11 Otsuka,M. ~ Konishi,S.~ Releaseof sul~tance Polikeimmunoreactivity from isolatedspinalcord of n~;wborn rat,Nature (Lond.),264 (1976) 83--84.
