Life Sciences, Vol. 46, pp. 1707-1714 Printed in the U.S.A.
NEUROPEPTIDE Gudrun
K IS P R E S E N T
Toresson,
Pergamon Press
IN HUMAN
CEREBROSPINAL
C a r o l a de las Carreras, Leif B e r t i l s s o n
Ernst
FLUID
Brodin
and
D e p a r t m e n t of C l i n i c a l P h a r m a c o l o g y at the K a r o l i n s k a Institute, H u d d i n g e Hospital, S-141 86 H u d d i n g e and D e p a r t m e n t of P h a r m a c o l o g y , K a r o l i n s k a Institute, S-I04 01 Stockholm, Sweden (Received in final form March 28, 1990) Summary Neurokinin A-like immunoreactivity (N~A-LI) in human c e r e b r o s p i n a l fluid (CSF) was d e t e r m i n e d by r a d i o i m m u n o assay (RIA) c o m b i n e d w i t h high p e r f o r m a n c e liquid chromatography (HPLC). The m a j o r i m m u n o r e a c t i v e c o m p o n e n t did not c o e l u t e w i t h NKA, but c o e l u t e d with n e u r o p e p t i d e K (NPK), w h i c h contains the N K A sequence in its C - t e r m i n u s . T r y p s i n t r e a t m e n t of this c o m p o n e n t from human CSF and of s y n t h e t i c NPK, p r o d u c e d a s u b s t a n c e w h i c h c o e l u t e d w i t h N K A in the HPLC system. W h e n the NKA-LI was o x i d i z e d w i t h h y d r o g e n p e r o x i d e and r e c h r o m a t o graphed, the i m m u n o r e a c t i v i t y c o e l u t e d w i t h NPK sulfoxide. The results indicate that the m a i n part of the NKA-LI in CSF is i d e n t i c a l w i t h NPK. The m e a n c o n c e n t r a t i o n of NPK m e a s u r e d in CSF from 6 h e a l t h y subjects by H P L C - R I A was 23 + ii (SD) pmol/L. N e u r o p e p t i d e s of the t a c h y k i n i n family have large s i m i l a r i t i e s in their C - t e r m i n a l a m i n o acid sequences. In a d d i t i o n to the m o s t w e l l k n o w n m e m b e r of this family, s u b s t a n c e P (SP), two other t a c h y k i n i n s have been i d e n t i f i e d in mammals, n a m e l y n e u r o k i n i n A (NKA) and n e u r o k i n i n B (NKB). Furthermore, two N - t e r m i n a l l y e x t e n d e d forms of NKA, i.e. n e u r o p e p t i d e K (NPK) (i) and r e c e n t l y n e u r o p e p t i d e y(NPT) (2) have been i s o l a t e d from m a m m a l i a n tissue.
Number of amblo acids
()
2'5
5'0
7'5
t
I
100
125
t.-I
I
~-preprotachykinin
I
I"KAI
Neuropeptide K FIG. Schematic precursor
1
s t r u c t u r e of 8 - p r e p r o t a c h y k i n i n , of SP, N K A and NPK.
the c o m m o n
0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
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SP and N K A have two c o m m o n p r e c u r s o r s , B- and y - p r e p r o t a c h y k i n i n s , shown by studies of the n u c l e o t i d e s e q u e n c e of c l o n e d cDNAs from b o v i n e (3) and rat b r a i n (4). The 8- and y - p r e p r o t a c h y k i n i n s c o n t a i n also the s e q u e n c e of N P K and NP7, r e s p e c t i v e l y (1,2). The s t r u c t u r e s of ~ - p r e p r o t a c h y k i n i n and N P K are s c h e m a t i c a l l y i l l u s t r a t e d in Fig 1. T h e r e is s u b s t a n t i a l e v i d e n c e that the t a c h y k i n i n s have s p e c i f i c roles as t r a n s m i t t e r s or n e u r o m o d u l a t o r s in v a r i o u s n e u r o n a l p a t h w a y s in the b r a i n and spinal c o r d (see 5,6). One p o s s i b l e a p p r o a c h to m o n i t o r the a c t i v i t y of c e n t r a l p e p t i d e r g i c pathways, w h i c h is a p p l i c a b l e also in humans, is to a n a l y z e the c o n t e n t of n e u r o p e p t i d e s in the c e r e b r o s p i n a l fluid (CSF). In a p r e v i o u s s t u d y we have shown by c o m b i n e d h i g h p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) - r a d i o i m m u n o a s s a y (RIA), that an N - t e r m i n a l l y e x t e m ~ e d form of SP is p r e s e n t and can be q u a n t i t a t e d in h u m a n CSF w h i l e SP is not p r e s e n t in d e t e c t a b l e c o n c e n t r a t i o n s (7). The p r e s e n t s t u d y was u n d e r t a k e n to i n v e s t i g a t e if N K A or r e l a t e d p e p t i d e s are p r e s e n t in h u m a n CSF. Materials
and M e t h o d s
S y n t h e t i c p e p t i d e s . NKA, NKB, NPK L a b o r a t o r i e s . The p e p t i d e c o n t e n t s o l u t i o n s w e r e v e r i f i e d by a m i n o NKA, N K B and NPK (NKA-ox, N K B - o x o b t a i n e d by t r e a t i n g the p e p t i d e s d e s c r i b e d below.
and NP7 w e r e from P e n i n s u l a of N K A and NPK in s t a n d a r d acid analysis. The s u l f o x i d e s of (dioxidized) and NPK-ox) w e r e w i t h h y d r o g e n p e r o x i d e (H202) as
C S F samples. CSF samples w e r e from h e a l t h y subjects. The lumbar p u n c t u r e s w e r e p e r f o r m e d w i t h a s t a n d a r d i z e d t e c h n i q u e (8). T w e l v e ml s a m p l e s w e r e c o l l e c t e d in p o l y p r o p y l e n e tubes, c e n t r i f u g e d and d i v i d e d into p o r t i o n s w h i c h w e r e frozen on dry ice. The samples w e r e then s t o r e d (less than three months) at -70°C until analysis. Chromatography. R e v e r s e - p h a s e HPLC was p e r f o r m e d u s i n g a S u p e l c o s i l L C - 1 8 - D B column, p a r t i c l e size 5~m, 15cm x 4 . 6 m m (Supelco). S a m p l e s of CSF (1-3 ml) w e r e i n j e c t e d into the HPLC s y s t e m via a p r e c o l u m n , 5 cm x 4.6 m m p a c k e d w i t h L i c h r o p r e p RP-18, p a r t i c l e size 25-40 pm (Merck). The samples w e r e e l u t e d w i t h a g r a d i e n t of a c e t o n i t r i l e (Lichrosolv, Merck) c o n t a i n i n g 0.08% t r i f l u o r o a c e t i c acid (TFA) (Uvasol, Merck) in w a t e r at a flow rate of 1 ml/min. The g r a d i e n t p r o f i l e is shown in Fig 2a. One ml f r a c t i o n s w e r e c o l l e c t e d and d r i e d in a v a c u u m c e n t r i f u g e b e f o r e RIA. The e l u t i o n p o s i t i o n s of NKA, NKB, NPK and t h e i r s u l f o x i d e forms and NPy w e r e d e t e r m i n e d by H P L C - R I A of CSF to w h i c h had b e e n a d d e d small a m o u n t s (4-100 fmol) of s y n t h e t i c p e p t i d e s . The r e c o v e r y of N K A from s a m p l e s c o n t a i n i n g 4.7 - 11.0 fmol was 89 + 8% (mean + SD; n=4). The r e c o v e r y of 15.1 fmol of N P K a d d e d to--2 ml samples of a r t i f i c i a l CSF and c a r r i e d t h r o u g h the e n t i r e a n a l y t i c a l p r o c e d u r e was 33 ~ 6% (mean ~ SD; n = 9). R a d i o i m m u n o assay. N K A - l i k e i m m u n o r e a c t i v i t y (NKA-LI) was d e t e r m i n e d w i t h an a n t i s e r u m (NKA 5) r a i s e d a g a i n s t N K A (9). The r e l a t i v e c r o s s - r e a c t i v i t y of this a n t i s e r u m w i t h o t h e r p e p t i d e s has been d e t e r m i n e d u s i n g N K A as a 100% r e f e r e n c e . The c r o s s r e a c t i v i t y w i t h N K A - o x was 72%, N K B 71%, N K B - o x 73%, k a s s i n i n 71%, e l e d o i s i n 60%, and w i t h SP, p h y s a l a e m i n , b o m b e s i n , n e u r o m e d i n B
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Neuropeptide K in Human CSF
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and n e u r o m e d i n C less than 0.1% in all cases (9). The crossr e a c t i v i t y of NPY was 86% (Brodin, u n p u b l i s h e d results). The c r o s s - r e a c t i v i t y w i t h N P K at the p r e s e n t assay c o n d i t i o n s was 37 + 2% (mean + SD; n=4). The a n t i s e r u m was used at a final d i l u t i o n of I:i00 0007 The radioligand, I z ~ I - ( T y r ° ) - N K A , w i t h a s p e c i f i c a c t i v i t y of about 1700 ~Ci/nmol was o b t a i n e d from MiLab, Malm~, Sweden. The RIA p r o c e d u r e was c a r r i e d out as d e s c r i b e d p r e v i o u s l y for the d e t e r m i n a t i o n of SP (7). The ICon (the amount of N K A needed to d i s p l a c e the r a d i o l i g a n d by 50~) using this m e t h o d was 3.1 fmol/100 ~i sample and the limit of d e t e c t i o n was 0.5 fmol/100 ~i sample. I n t e r - a s s a y c o e f f i c i e n t s of variation, d e t e r m i n e d by a n a l y s i n g frozen a l i q u o t s of a r t i f i c i a l CSF c o n t a i n i n g s y n t h e t i c NKA at a c o n c e n t r a t i o n of 1.2 and 5.5 fmol/100 ~i sample w e r e 13% and 12%, r e s p e c t i v e l y (n=8). The c o r r e s p o n d i n g values o b t a i n e d using samples (I00 ~i) c o n t a i n i n g 6.9 and 13.7 fmol NPK, w e r e 17% and 12%, r e s p e c t i v e l y (n=6). Oxidation. S y n t h e t i c p e p t i d e s and e v a p o r a t e d HPLC fractions after s e p a r a t i o n of CSF c o m p o n e n t s were d i s s o l v e d in 1 ml of 0.01 M acetic acid c o n t a i n i n g 0.5% H^O 2. The samples w e r e kept in room t e m p e r a t u r e for one hour and ~hen e v a p o r a t e d to d r y n e s s in a v a c u u m centrifuge. The dried samples were d i s s o l v e d in 50 ~i 45% C H ~ C N in 0.08% T F A and d i l u t e d to 1 ml w i t h a r t i f i c i a l CSF before J they w e r e i n j e c t e d into the HPLC-system.
a
10
NKB-ox, NPK-ox, NKA-ox
A
NKA
t
o
"-"
*
, ~ 0 1J
t
ii
......
o
25
~--
-_.
U
......... !
o E
0
E
10
o
NKB NPK
t
,
-
lO
-
20
30
40 b (Trypsin)
i
Z
0
10
FIG.
2'0 3'0 Fraction number
410
2
HPLC of two ml of CSF from a h e a l t h y human subject (a). F r a c t i o n s 29-31 w e r e c o l l e c t e d and treated w i t h trypsin. The c h r o m a t o g r a m of the r e i n j e c t e d trypsin treated sample is shown in (b). The samples w e r e eluted w i t h an a c e t o n i t r i l e (CH~CN) g r a d i e n t as i n d i c a t e d in (a). The arrows show the r e t e n t i o n times of s y n t h e t i c peptides.
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Neuropeptide K in Human CSF
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T r y p s i n i z a t i o n . E v a p o r a t e d HPLC f r a c t i o n s were i n c u b a t e d w i t h 5 ~g t r y p s i n (TPCK t r e a t e d A Sigma) in 200 ~i 0.1 M a m m o n i u m b i c a r b o n a t e b u f f e r (pH 7.8) at 37vC for 4 hours. The r e a c t i o n was t e r m i n a t e d by a d d i n g 25 ~i of 2 M a c e t i c acid and h e a t i n g the samples to 100°C for 5 min. The i n c u b a t i o n m i x t u r e was d i l u t e d w i t h 1 ml a r t i f i c i a l CSF b e f o r e the s a m p l e s w e r e i n j e c t e d o n t o the p r e c o l u m n of the HPLC system. E x p e r i m e n t s w h e r e s y n t h e t i c NPK was t r e a t e d the same w a y w e r e also p e r f o r m e d . Results H u m a n CSF s a m p l e s from 6 h e a l t h y s u b j e c t s w e r e a n a l y s e d by c o m b i n e d H P L C - R I A . In all samples the m a j o r c o m p o n e n t s of NKA-LI a p p e a r e d in f r a c t i o n s that c o r r e s p o n d e d to the e l u t i o n p o s i t i o n of NPK w h i l e the a m o u n t of i m m u n o r e a c t i v i t y in the p o s i t i o n of N K A and NP¥ (which a l m o s t coeluted) was c o m p a r a t i v e l y small and no i m m u n o r e a c t i v i t y was found at the p o s i t i o n of NKB (Figs 2a, 3a). In some of the s a m p l e s m i n o r i m m u n o r e a c t i v e c o m p o n e n t s that e l u t e d after NPK c o u l d also be d e t e c t e d (Figs 2a, 3a). In CSF from 6 h e a l t h y s u b j e c t s the c o n c e n t r a t i o n of i m m u n o r e a c t i v i t y e l u t e d at the r e t e n t i o n time of NPK, e x p r e s s e d in N K A e q u i v a l e n t s , was 2.8 + 1.4 p m o l / L (mean + SD). As N P K has a m e a n r e c o v e r y through the a n a l y t i c a l p r o c e d u r e of 33% and a c r o s s r e a c t i v i t y in RIA of 37% (see M a t e r i a l s and M e t h o d s ) , the c o n c e n t r a t i o n of NPK in CSF of these 6 s u b j e c t s c o u l d be e s t i m a t e d to 23 + ii pmol/L.
NPK-ox
NPK t
10
NKB-ox t
A t-
o
NKA-ox
NKA t
5
NKB t
t
NP¥
m
L.
o
E
1'0
0
2'0
40
3'0
®
b
(Hz02) !
z
0
1'0
FIG.
2'0 3'0 F r a c t i o n number
40
3
H P L C of three ml of p o o l e d C S F from two h e a l t h y human s u b j e c t s (a). F r a c t i o n s 23-25 in (a) w e r e o x i d i z e d w i t h H?O? and r e c h r o m a t o g r a p h e d (b). The r e t e n t i o n times of s y n t h e t i c p e p t i d e s are i n d i c a t e d in a. The e l u t i o n c o n d i t i o n s were the same as in Fig. 2, but a n o t h e r column was used.
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Neuropeptide K in Human CSF
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The two H P L C c o l u m n s ( S u p e l c o s i l LC-18-DB) u s e d in the p r e s e n t study gave d i f f e r e n t r e t e n t i o n times of s y n t h e t i c N P K and N P K - o x (compare Figs 2a and 3a) w h i l e s y n t h e t i c NKA, NKB, N K A - o x and N K B - o x w e r e e l u t e d at i d e n t i c a l p o s i t i o n (see Figs 2a and 3a). However, d e s p i t e this i n t e r c o l u m n v a r i a b i l i t y the m a i n c o m p o n e n t of N K A - L I in h u m a n C S F c o e l u t e d w i t h s y n t h e t i c NPK on both c o l u m n s S e r i a l d i l u t i o n of the i m m u n o r e a c t i v e c o m p o n e n t from CSF, w h i c h c o e l u t e d w i t h N P K on HPLC, p r o d u c e d a curve that was p a r a l l e l to the d i s p l a c e m e n t c u r v e s of s y n t h e t i c N K A and N P K (Fig 4). of CSF
Dilution
B/B0
1/641/32 1/161/8 L
i
i
i
sample
1/4
1/2
1/1
i
i
r
1.0-
0.5 NKA-LIin CSF
0.O
o11
-
1'o
~
1'o
.
lbO
Femtomol of NKA or NPK FIG.
4
D i l u t i o n c u r v e s of s y n t h e t i c NKA, NPK and of NKA-LI from h u m a n CSF d e t e r m i n e d w i t h RIA. B/B r e p r e s e n t s the f r a c t i o n of r a d i o a c t i v i t y b o u n d to the antiserum. B= r a d i o a c t i v i t y in samples, B = r a d i o a c t i v i t y in zero sample. o To f u r t h e r c h a r a c t e r i z e this form of N K A - L I in CSF, HPLC f r a c t i o n s 29-31 (see Fig 2a) w e r e c o l l e c t e d and i n c u b a t e d w i t h trypsin. Rec h r o m a t o g r a p h y and s u b s e q u e n t RIA of the sample r e v e a l e d a single c o m p o n e n t at the e l u t i o n p o s i t i o n of N K A (Fig 2b). S i m i l a r l y t r y p s i n d i g e s t i o n of s y n t h e t i c N P K p r o d u c e d a c o m p o n e n t w h i c h c o e l u t e d w i t h NKA. To i n v e s t i g a t e if o x i d a t i o n also a f f e c t e d the m o b i l i t y of the i m m u n o r e a c t i v e c o m p o n e n t in CSF in the same w a y as s y n t h e t i c NPK, the d r i e d IIPLC f r a c t i o n s 23-25 (Fig 3a) w e r e i n c u b a t e d w i t h H^O 2 (see m e t h o d s ) . W h e n the o x i d i z e d sample was r e i n j e c t e d into t~e H P L C - s y s t e m , the i m m u n o r e a c t i v i t y was e l u t e d s o m e w h a t e a r l i e r than NPK and a p p e a r e d n o w at the p o s i t i o n of o x i d i z e d N P K (Fig 3b).
Discussion HPLC c h a r a c t e r i z a t i o n of N K A - L I in human CSF r e v e a l e d that m o s t of the i m m u n o r e a c t i v i t y c o e l u t e d w i t h NPK. T r y p s i n t r e a t m e n t of this
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Neuropeptide K in Human CSF
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compound yielded NKA-LI coeluting with synthetic NKA which i n d i c a t e s that the i m m u n o r e a c t i v e c o m p o u n d c o n t a i n s the N K A sequence. It has p r e v i o u s l y been r e p o r t e d that NPK i s o l a t e d from p o r c i n e b r a i n p r o d u c e N K A u p o n t r y p s i n d i g e s t i o n (i). F u r t h e r m o r e , w h e n the N K A - L I from CSF was o x i d i z e d and r e c h r o m a t o g r a p h e d the i m m u n o r e a c t i v i t y was found at the p o s i t i o n of NPK-ox, w h i c h elutes s l i g h t l y e a r l i e r than NPK. P r e v i o u s l y , NPK has b e e n r e p o r t e d to elute b e t w e e n N K A and NKB upon r e v e r s e - p h a s e HPLC (10-14). In one r e p o r t (15), however, N P K was found to elute a f t e r NKB. The o b s e r v a t i o n in the p r e s e n t study that the r e t e n t i o n time of N P K and N P K - o x (but not of the o t h e r t a c h y k i n i n s ) w e r e d i f f e r e n t u s i n g two a p p a r e n t l y i d e n t i c a l c o l u m n s u n d e r i d e n t i c a l e l u t i o n c o n d i t i o n s was s u r p r i s i n g e s p e c i a l l y since it r e s u l t e d in d i f f e r e n t e l u t i o n o r d e r s of NPK and NKB. The r e t e n t i o n of c o m p o u n d s on r e v e r s e - p h a s e c o l u m n s is h o w e v e r c o n s i d e r e d to be due to a m i x e d r e t e n t i o n m e c h a n i s m and it is thus p o s s i b l e that small c o l u m n - to c o l u m n d i f f e r e n c e s of the s t a t i o n a r y p h a s e can cause a c h a n g e of the e l u t i o n o r d e r of s u b s t a n c e s that elute r a t h e r close to each o t h e r (as e.g. N P K and N K B in the p r e s e n t study). The m a i n N K A - L I c o m p o n e n t in CSF also had d i f f e r e n t r e t e n t i o n times on the two c o l u m n s but c o e l u t e d always w i t h s y n t h e t i c NPK, also i n d i c a t i n g that the s u b s t a n c e is i n d e e d i d e n t i c a l to NPK. In a d d i t i o n to NPK, a n o t h e r N - t e r m i n a l l y e x t e n d e d form of NKA, NPY, has r e c e n t l y been i d e n t i f i e d in m a m m a l i a n t i s s u e s (2). The N K A a n t i s e r u m , N K A 5, reacts also w i t h NP7. However, this p e p t i d e e l u t e d at m u c h lower c o n c e n t r a t i o n s of a c e t o n i t r i l e than NPK and a p p e a r s c l o s e to the p o s i t i o n of NKA. We c o u l d t h e r e f o r e e x c l u d e that the m a j o r c o m p o n e n t of N K A - L I in CSF is NPy. F r o m the p r e s e n t r e s u l t s we c o n c l u d e that the c o m p o u n d r e s p o n s i b l e for the m a i n N K A - L I in CSF is m o s t l i k e l y to be NPK. The c o n c e n t r a t i o n s of N K A - L I in CSF from h e a l t h y s u b j e c t s w e r e m e a s u r e d by H P L C - R I A u s i n g N K A as standard. E x p r e s s e d in NPK e q u i v a l e n t s , o b t a i n e d by c o r r e c t i n g the r e s u l t s for c r o s s - r e a c t i v i t y and r e c o v e r y of N P K (see M a t e r i a l s and Methods) the c o n c e n t r a t i o n was c a l c u l a t e d to be 23 + ii p m o l / L (mean + SD; n=6). This transf o r m a t i o n seems r e a s o n a b l e since the d T s p l a c e m e n t c u r v e s of N K A and N P K w e r e p a r a l l e l . The H P L C - R I A m e t h o d was d e v e l o p e d for the a n a l y s i s p r i m a r i l y of N K A and we have a h i g h r e c o v e r y of this peptide. The low but r e p r o d u c i b l e r e c o v e r y of 33 + 6% t h r o u g h the e n t i r e a n a l y t i c a l p r o c e d u r e for N P K is p r o b a b l y due to a b s o r p t i o n to v a r i o u s c o m p o n e n t s of the a n a l y t i c a l system. M a c D o n a l d et al (16) have d o c u m e n t e d the p r o b l e m s w i t h N P K and a b s o r p t i o n . N P K has p r e v i o u s l y been found to be one of the m a j o r t a c h y k i n i n s in p l a s m a and in a c e t i c acid e x t r a c t s of tumor tissue from c a r c i n o i d p a t i e n t s (I0). N P K has b i o l o g i c a l a c t i v i t y in several s y s t e m s and T a t e m o t o et al (i) have s u g g e s t e d that NPK m i g h t be a n e u r o m e s s e n g e r in i t s e l f and not just a p r e c u r s o r of NKA. This h y p o t h e s i s is s t r e n g t h e n e d by the r e s u l t s of D i e z - G u e r r a et al (17) w h i c h shows that NPK can be r e l e a s e d by d e p o l a r i z i n g stimuli from rat s u b s t a n t i a nigra. It is thus p o s s i b l e that NPK in CSF m a y be used as a m a r k e r for the a c t i v i t y of c e n t r a l N P K - c o n t a i n i n g neurons.
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We have r e c e n t l y d e m o n s t r a t e d that human CSF contains also an N - t e r m i n a l l y e x t e n d e d form of SP, w h i l e (undeca) SP itself could not be d e t e c t e d (7). A l s o this e x t e n d e d t a c h y k i n i n m o l e c u l e is r e l e a s e d from rat C N S - t i s s u e in vitro by d e p o l a r i z i n g stimuli s u g g e s t i n g a n e u r o m e s s e n g e r role (18). In a d d i t i o n we have o b t a i n e d data s u g g e s t i n g that SP e x t e n d e d both in the C- and N - t e r m i n a l ends is p r e s e n t in CSF (7). Thus, it seems as if the larger m o l e c u l a r forms of t a c h y k i n i n s rather than the "classical" ones are p r e s e n t in human CSF. One r e a s o n for this could be that the e x t e n d e d c o m p a r e d w i t h the smaller t a c h y k i n i n s are more m e t a b o l i c a l l y stable in the extrac e l l u l a r space or in the CSF. This subject o b v i o u s l y deserves further i n v e s t i g a t i o n . However, it has been r e p o r t e d that the p l a s m a half life of NPK is longer than that of N K A when these p e p t i d e s are infused i n t r a v e n o u s l y in guinea pigs (19). A h i g h r e s i s t a n c e of NPK to d e g r a d a t i o n w o u l d make it less likely to have a role as a transmitter, but it is also p o s s i b l e that NPK has long term effects d i f f e r e n t from a c l a s s i c a l transmitter. In p a r a l l e l to our own findings on CSF tachykinins, N y b e r g and c o l l a b o r a t o r s have r e p o r t e d that there is more of high than of low m o l e c u l a r w e i g h t o p i o i d p e p t i d e s in human CSF, p o s s i b l y due to a higher r e s i s t a n c e of the larger m o l e c u l e s to d e g r a d i n g a c t i v i t y (20,21). In c o n c l u s i o n our results d e m o n s t r a t e the p r e s e n c e of NPK in human CSF, while N K A and NKB do not seem to be present, or occur at m u c h lower c o n c e n t r a t i o n s . The p o s s i b l e u s e f u l n e s s of NPK in CSF as a m a r k e r for the a c t i v i t y in central N P K - c o n t a i n i n g neurons d e s e r v e s investigation. Acknowledgements F i n a n c i a l support was given by the S w e d i s h M e d i c a l R e s e a r c h C o u n c i l (8270, 6836), the Bank of Sweden T e r c e n t e n a r y F o u n d a t i o n (83/38), the W a l l e n b e r g F o u n d a t i o n and the K a r o l i n s k a Institute. P r o f e s s o r Hans J~rnvall, D e p a r t m e n t of C h e m i s t r y I, K a r o l i n s k a I n s t i t u t e is g r a t e f u l l y a c k n o w l e d g e d for help with the amino acid determinations.
References i. 2. 3. 4. 5. 6. 7. 8.
K. TATEMOTO, J.M, LUNDBERG, H. J O R N V A L L and V. MUTT, B i o c h e m B i o p h y s Res C o m m 128, 947-953 (1985) R. KAGE, G.P. McGREGOR, L. T H I M and J.M. CONLON, J N e u r o c h e m 50, 1412-1417 (1988) H. NAWA, T, HIROSE, H. TAKASHIMA, S. I N A Y A M A and S. N A K A N I S H I N a t u r e 306, 32-36 (1983) J.E. KRAUSE, J.M. CHIRGWIN, M.S. CARTER, Z.S. XU and A.D. HERSHEY, Proc Natl A c a d Sci 84, 881-885 (1987) B. PERNOW, Pharmacol. Rev. 35, 85-141 (1983) J.E. MAGGIO, Ann. Rev. Neurosci. Ii, 13-28 (1988) G. TORESSON, E. BRODIN, A. WAHLSTR--~M and L. BERTILSSON, J N e u r o c h e m 50, 1701-1707 (1988). L. B E R T I L S S O N and M. ASBERG, in F r o n t i e r s in B i o c h e m i c a l and P h a r m a c o l o g i c a l R e s e a r c h in Depression, E. Usdin, M. Asberg, L. B e r t i l s s o n and F. sj~qvist, eds, pp 27-34 Raven Press, N e w York (1984).
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9. 10.
II. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21.
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E. BRODIN, N. LINDEFORS, C-J. DALSGAARD, E. T H E O D O R S S O N N O R H E I M and S. ROSELL, Regul Pept 13, 253-272 (1986) E. T H E O D O R S S O N - N O R H E I M , I. NORHEIM, K. ~BERG, E. BRODIN, J.M. LUNDBERG, K. T A T E M O T O and P.G. LINDGREN, B i o c h e m B i o p h y s Res Comm 131, 77-83 (1985). H. ARAI and P.C. EMSON, Brain R e s e a r c h 399, 240-249 (1986). C.F. DEACON, D.V. AGOSTON, R. N A U and J.M CONLON, J. Neurochem. 48, 141-146 (1987). H.-P. TOO, J.L. C O R D O V A and J.E. MAGGIO, P e p t i d e s 10, 25-30 (1988). B. B E D I N G - B A R N E K O W and E. BRODIN, Regul. Peptides 25, 199-206 (1989) . K. TATEISHI, Y. M A T S O U K A and T. HAMAOKA, Reg. Peptides 24, 245-257 (1989). M . R . M A C D O N A L D , J.TAKEDA, C.M. RICE and J.E. KRAUSE, J Bio C h e m 264, 15578-15592 (1989). F.J. DIEZ-GUERRA, D.J.S. S I R I N A T H S I N G H J I and P.C. EMSON, N e u r o s c i e n c e 27, 527-536 (1988). G. TORESSON, C. DE LAS CARRERAS, L. B E R T I L S S O N and E. BRODIN, Reg. P e p t i d e s (In press). C-L. MARTLING, E. T H E O D O R S S O N - N O R H E I M , I . N O R H E I M and J.M. LUNDBERG, N a u n y n - S c h m i e d e b e r g ' s A r c h P h a r m a c o l 336, 183-189 (1987). F. N Y B E R G and L. TERENIUS, N e u r o p e p t i d e s ~, 537-540 (1985). F. NYBERG, I. N Y L A N D E R and L. TERENIUS, Brain R e s e a r c h 371, 278-286 (1986).
NEUROPEPTIDE Gudrun
K IS P R E S E N T
Toresson,
Pergamon Press
IN HUMAN
CEREBROSPINAL
C a r o l a de las Carreras, Leif B e r t i l s s o n
Ernst
FLUID
Brodin
and
D e p a r t m e n t of C l i n i c a l P h a r m a c o l o g y at the K a r o l i n s k a Institute, H u d d i n g e Hospital, S-141 86 H u d d i n g e and D e p a r t m e n t of P h a r m a c o l o g y , K a r o l i n s k a Institute, S-I04 01 Stockholm, Sweden (Received in final form March 28, 1990) Summary Neurokinin A-like immunoreactivity (N~A-LI) in human c e r e b r o s p i n a l fluid (CSF) was d e t e r m i n e d by r a d i o i m m u n o assay (RIA) c o m b i n e d w i t h high p e r f o r m a n c e liquid chromatography (HPLC). The m a j o r i m m u n o r e a c t i v e c o m p o n e n t did not c o e l u t e w i t h NKA, but c o e l u t e d with n e u r o p e p t i d e K (NPK), w h i c h contains the N K A sequence in its C - t e r m i n u s . T r y p s i n t r e a t m e n t of this c o m p o n e n t from human CSF and of s y n t h e t i c NPK, p r o d u c e d a s u b s t a n c e w h i c h c o e l u t e d w i t h N K A in the HPLC system. W h e n the NKA-LI was o x i d i z e d w i t h h y d r o g e n p e r o x i d e and r e c h r o m a t o graphed, the i m m u n o r e a c t i v i t y c o e l u t e d w i t h NPK sulfoxide. The results indicate that the m a i n part of the NKA-LI in CSF is i d e n t i c a l w i t h NPK. The m e a n c o n c e n t r a t i o n of NPK m e a s u r e d in CSF from 6 h e a l t h y subjects by H P L C - R I A was 23 + ii (SD) pmol/L. N e u r o p e p t i d e s of the t a c h y k i n i n family have large s i m i l a r i t i e s in their C - t e r m i n a l a m i n o acid sequences. In a d d i t i o n to the m o s t w e l l k n o w n m e m b e r of this family, s u b s t a n c e P (SP), two other t a c h y k i n i n s have been i d e n t i f i e d in mammals, n a m e l y n e u r o k i n i n A (NKA) and n e u r o k i n i n B (NKB). Furthermore, two N - t e r m i n a l l y e x t e n d e d forms of NKA, i.e. n e u r o p e p t i d e K (NPK) (i) and r e c e n t l y n e u r o p e p t i d e y(NPT) (2) have been i s o l a t e d from m a m m a l i a n tissue.
Number of amblo acids
()
2'5
5'0
7'5
t
I
100
125
t.-I
I
~-preprotachykinin
I
I"KAI
Neuropeptide K FIG. Schematic precursor
1
s t r u c t u r e of 8 - p r e p r o t a c h y k i n i n , of SP, N K A and NPK.
the c o m m o n
0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
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SP and N K A have two c o m m o n p r e c u r s o r s , B- and y - p r e p r o t a c h y k i n i n s , shown by studies of the n u c l e o t i d e s e q u e n c e of c l o n e d cDNAs from b o v i n e (3) and rat b r a i n (4). The 8- and y - p r e p r o t a c h y k i n i n s c o n t a i n also the s e q u e n c e of N P K and NP7, r e s p e c t i v e l y (1,2). The s t r u c t u r e s of ~ - p r e p r o t a c h y k i n i n and N P K are s c h e m a t i c a l l y i l l u s t r a t e d in Fig 1. T h e r e is s u b s t a n t i a l e v i d e n c e that the t a c h y k i n i n s have s p e c i f i c roles as t r a n s m i t t e r s or n e u r o m o d u l a t o r s in v a r i o u s n e u r o n a l p a t h w a y s in the b r a i n and spinal c o r d (see 5,6). One p o s s i b l e a p p r o a c h to m o n i t o r the a c t i v i t y of c e n t r a l p e p t i d e r g i c pathways, w h i c h is a p p l i c a b l e also in humans, is to a n a l y z e the c o n t e n t of n e u r o p e p t i d e s in the c e r e b r o s p i n a l fluid (CSF). In a p r e v i o u s s t u d y we have shown by c o m b i n e d h i g h p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) - r a d i o i m m u n o a s s a y (RIA), that an N - t e r m i n a l l y e x t e m ~ e d form of SP is p r e s e n t and can be q u a n t i t a t e d in h u m a n CSF w h i l e SP is not p r e s e n t in d e t e c t a b l e c o n c e n t r a t i o n s (7). The p r e s e n t s t u d y was u n d e r t a k e n to i n v e s t i g a t e if N K A or r e l a t e d p e p t i d e s are p r e s e n t in h u m a n CSF. Materials
and M e t h o d s
S y n t h e t i c p e p t i d e s . NKA, NKB, NPK L a b o r a t o r i e s . The p e p t i d e c o n t e n t s o l u t i o n s w e r e v e r i f i e d by a m i n o NKA, N K B and NPK (NKA-ox, N K B - o x o b t a i n e d by t r e a t i n g the p e p t i d e s d e s c r i b e d below.
and NP7 w e r e from P e n i n s u l a of N K A and NPK in s t a n d a r d acid analysis. The s u l f o x i d e s of (dioxidized) and NPK-ox) w e r e w i t h h y d r o g e n p e r o x i d e (H202) as
C S F samples. CSF samples w e r e from h e a l t h y subjects. The lumbar p u n c t u r e s w e r e p e r f o r m e d w i t h a s t a n d a r d i z e d t e c h n i q u e (8). T w e l v e ml s a m p l e s w e r e c o l l e c t e d in p o l y p r o p y l e n e tubes, c e n t r i f u g e d and d i v i d e d into p o r t i o n s w h i c h w e r e frozen on dry ice. The samples w e r e then s t o r e d (less than three months) at -70°C until analysis. Chromatography. R e v e r s e - p h a s e HPLC was p e r f o r m e d u s i n g a S u p e l c o s i l L C - 1 8 - D B column, p a r t i c l e size 5~m, 15cm x 4 . 6 m m (Supelco). S a m p l e s of CSF (1-3 ml) w e r e i n j e c t e d into the HPLC s y s t e m via a p r e c o l u m n , 5 cm x 4.6 m m p a c k e d w i t h L i c h r o p r e p RP-18, p a r t i c l e size 25-40 pm (Merck). The samples w e r e e l u t e d w i t h a g r a d i e n t of a c e t o n i t r i l e (Lichrosolv, Merck) c o n t a i n i n g 0.08% t r i f l u o r o a c e t i c acid (TFA) (Uvasol, Merck) in w a t e r at a flow rate of 1 ml/min. The g r a d i e n t p r o f i l e is shown in Fig 2a. One ml f r a c t i o n s w e r e c o l l e c t e d and d r i e d in a v a c u u m c e n t r i f u g e b e f o r e RIA. The e l u t i o n p o s i t i o n s of NKA, NKB, NPK and t h e i r s u l f o x i d e forms and NPy w e r e d e t e r m i n e d by H P L C - R I A of CSF to w h i c h had b e e n a d d e d small a m o u n t s (4-100 fmol) of s y n t h e t i c p e p t i d e s . The r e c o v e r y of N K A from s a m p l e s c o n t a i n i n g 4.7 - 11.0 fmol was 89 + 8% (mean + SD; n=4). The r e c o v e r y of 15.1 fmol of N P K a d d e d to--2 ml samples of a r t i f i c i a l CSF and c a r r i e d t h r o u g h the e n t i r e a n a l y t i c a l p r o c e d u r e was 33 ~ 6% (mean ~ SD; n = 9). R a d i o i m m u n o assay. N K A - l i k e i m m u n o r e a c t i v i t y (NKA-LI) was d e t e r m i n e d w i t h an a n t i s e r u m (NKA 5) r a i s e d a g a i n s t N K A (9). The r e l a t i v e c r o s s - r e a c t i v i t y of this a n t i s e r u m w i t h o t h e r p e p t i d e s has been d e t e r m i n e d u s i n g N K A as a 100% r e f e r e n c e . The c r o s s r e a c t i v i t y w i t h N K A - o x was 72%, N K B 71%, N K B - o x 73%, k a s s i n i n 71%, e l e d o i s i n 60%, and w i t h SP, p h y s a l a e m i n , b o m b e s i n , n e u r o m e d i n B
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and n e u r o m e d i n C less than 0.1% in all cases (9). The crossr e a c t i v i t y of NPY was 86% (Brodin, u n p u b l i s h e d results). The c r o s s - r e a c t i v i t y w i t h N P K at the p r e s e n t assay c o n d i t i o n s was 37 + 2% (mean + SD; n=4). The a n t i s e r u m was used at a final d i l u t i o n of I:i00 0007 The radioligand, I z ~ I - ( T y r ° ) - N K A , w i t h a s p e c i f i c a c t i v i t y of about 1700 ~Ci/nmol was o b t a i n e d from MiLab, Malm~, Sweden. The RIA p r o c e d u r e was c a r r i e d out as d e s c r i b e d p r e v i o u s l y for the d e t e r m i n a t i o n of SP (7). The ICon (the amount of N K A needed to d i s p l a c e the r a d i o l i g a n d by 50~) using this m e t h o d was 3.1 fmol/100 ~i sample and the limit of d e t e c t i o n was 0.5 fmol/100 ~i sample. I n t e r - a s s a y c o e f f i c i e n t s of variation, d e t e r m i n e d by a n a l y s i n g frozen a l i q u o t s of a r t i f i c i a l CSF c o n t a i n i n g s y n t h e t i c NKA at a c o n c e n t r a t i o n of 1.2 and 5.5 fmol/100 ~i sample w e r e 13% and 12%, r e s p e c t i v e l y (n=8). The c o r r e s p o n d i n g values o b t a i n e d using samples (I00 ~i) c o n t a i n i n g 6.9 and 13.7 fmol NPK, w e r e 17% and 12%, r e s p e c t i v e l y (n=6). Oxidation. S y n t h e t i c p e p t i d e s and e v a p o r a t e d HPLC fractions after s e p a r a t i o n of CSF c o m p o n e n t s were d i s s o l v e d in 1 ml of 0.01 M acetic acid c o n t a i n i n g 0.5% H^O 2. The samples w e r e kept in room t e m p e r a t u r e for one hour and ~hen e v a p o r a t e d to d r y n e s s in a v a c u u m centrifuge. The dried samples were d i s s o l v e d in 50 ~i 45% C H ~ C N in 0.08% T F A and d i l u t e d to 1 ml w i t h a r t i f i c i a l CSF before J they w e r e i n j e c t e d into the HPLC-system.
a
10
NKB-ox, NPK-ox, NKA-ox
A
NKA
t
o
"-"
*
, ~ 0 1J
t
ii
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o
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~--
-_.
U
......... !
o E
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E
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o
NKB NPK
t
,
-
lO
-
20
30
40 b (Trypsin)
i
Z
0
10
FIG.
2'0 3'0 Fraction number
410
2
HPLC of two ml of CSF from a h e a l t h y human subject (a). F r a c t i o n s 29-31 w e r e c o l l e c t e d and treated w i t h trypsin. The c h r o m a t o g r a m of the r e i n j e c t e d trypsin treated sample is shown in (b). The samples w e r e eluted w i t h an a c e t o n i t r i l e (CH~CN) g r a d i e n t as i n d i c a t e d in (a). The arrows show the r e t e n t i o n times of s y n t h e t i c peptides.
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T r y p s i n i z a t i o n . E v a p o r a t e d HPLC f r a c t i o n s were i n c u b a t e d w i t h 5 ~g t r y p s i n (TPCK t r e a t e d A Sigma) in 200 ~i 0.1 M a m m o n i u m b i c a r b o n a t e b u f f e r (pH 7.8) at 37vC for 4 hours. The r e a c t i o n was t e r m i n a t e d by a d d i n g 25 ~i of 2 M a c e t i c acid and h e a t i n g the samples to 100°C for 5 min. The i n c u b a t i o n m i x t u r e was d i l u t e d w i t h 1 ml a r t i f i c i a l CSF b e f o r e the s a m p l e s w e r e i n j e c t e d o n t o the p r e c o l u m n of the HPLC system. E x p e r i m e n t s w h e r e s y n t h e t i c NPK was t r e a t e d the same w a y w e r e also p e r f o r m e d . Results H u m a n CSF s a m p l e s from 6 h e a l t h y s u b j e c t s w e r e a n a l y s e d by c o m b i n e d H P L C - R I A . In all samples the m a j o r c o m p o n e n t s of NKA-LI a p p e a r e d in f r a c t i o n s that c o r r e s p o n d e d to the e l u t i o n p o s i t i o n of NPK w h i l e the a m o u n t of i m m u n o r e a c t i v i t y in the p o s i t i o n of N K A and NP¥ (which a l m o s t coeluted) was c o m p a r a t i v e l y small and no i m m u n o r e a c t i v i t y was found at the p o s i t i o n of NKB (Figs 2a, 3a). In some of the s a m p l e s m i n o r i m m u n o r e a c t i v e c o m p o n e n t s that e l u t e d after NPK c o u l d also be d e t e c t e d (Figs 2a, 3a). In CSF from 6 h e a l t h y s u b j e c t s the c o n c e n t r a t i o n of i m m u n o r e a c t i v i t y e l u t e d at the r e t e n t i o n time of NPK, e x p r e s s e d in N K A e q u i v a l e n t s , was 2.8 + 1.4 p m o l / L (mean + SD). As N P K has a m e a n r e c o v e r y through the a n a l y t i c a l p r o c e d u r e of 33% and a c r o s s r e a c t i v i t y in RIA of 37% (see M a t e r i a l s and M e t h o d s ) , the c o n c e n t r a t i o n of NPK in CSF of these 6 s u b j e c t s c o u l d be e s t i m a t e d to 23 + ii pmol/L.
NPK-ox
NPK t
10
NKB-ox t
A t-
o
NKA-ox
NKA t
5
NKB t
t
NP¥
m
L.
o
E
1'0
0
2'0
40
3'0
®
b
(Hz02) !
z
0
1'0
FIG.
2'0 3'0 F r a c t i o n number
40
3
H P L C of three ml of p o o l e d C S F from two h e a l t h y human s u b j e c t s (a). F r a c t i o n s 23-25 in (a) w e r e o x i d i z e d w i t h H?O? and r e c h r o m a t o g r a p h e d (b). The r e t e n t i o n times of s y n t h e t i c p e p t i d e s are i n d i c a t e d in a. The e l u t i o n c o n d i t i o n s were the same as in Fig. 2, but a n o t h e r column was used.
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Neuropeptide K in Human CSF
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The two H P L C c o l u m n s ( S u p e l c o s i l LC-18-DB) u s e d in the p r e s e n t study gave d i f f e r e n t r e t e n t i o n times of s y n t h e t i c N P K and N P K - o x (compare Figs 2a and 3a) w h i l e s y n t h e t i c NKA, NKB, N K A - o x and N K B - o x w e r e e l u t e d at i d e n t i c a l p o s i t i o n (see Figs 2a and 3a). However, d e s p i t e this i n t e r c o l u m n v a r i a b i l i t y the m a i n c o m p o n e n t of N K A - L I in h u m a n C S F c o e l u t e d w i t h s y n t h e t i c NPK on both c o l u m n s S e r i a l d i l u t i o n of the i m m u n o r e a c t i v e c o m p o n e n t from CSF, w h i c h c o e l u t e d w i t h N P K on HPLC, p r o d u c e d a curve that was p a r a l l e l to the d i s p l a c e m e n t c u r v e s of s y n t h e t i c N K A and N P K (Fig 4). of CSF
Dilution
B/B0
1/641/32 1/161/8 L
i
i
i
sample
1/4
1/2
1/1
i
i
r
1.0-
0.5 NKA-LIin CSF
0.O
o11
-
1'o
~
1'o
.
lbO
Femtomol of NKA or NPK FIG.
4
D i l u t i o n c u r v e s of s y n t h e t i c NKA, NPK and of NKA-LI from h u m a n CSF d e t e r m i n e d w i t h RIA. B/B r e p r e s e n t s the f r a c t i o n of r a d i o a c t i v i t y b o u n d to the antiserum. B= r a d i o a c t i v i t y in samples, B = r a d i o a c t i v i t y in zero sample. o To f u r t h e r c h a r a c t e r i z e this form of N K A - L I in CSF, HPLC f r a c t i o n s 29-31 (see Fig 2a) w e r e c o l l e c t e d and i n c u b a t e d w i t h trypsin. Rec h r o m a t o g r a p h y and s u b s e q u e n t RIA of the sample r e v e a l e d a single c o m p o n e n t at the e l u t i o n p o s i t i o n of N K A (Fig 2b). S i m i l a r l y t r y p s i n d i g e s t i o n of s y n t h e t i c N P K p r o d u c e d a c o m p o n e n t w h i c h c o e l u t e d w i t h NKA. To i n v e s t i g a t e if o x i d a t i o n also a f f e c t e d the m o b i l i t y of the i m m u n o r e a c t i v e c o m p o n e n t in CSF in the same w a y as s y n t h e t i c NPK, the d r i e d IIPLC f r a c t i o n s 23-25 (Fig 3a) w e r e i n c u b a t e d w i t h H^O 2 (see m e t h o d s ) . W h e n the o x i d i z e d sample was r e i n j e c t e d into t~e H P L C - s y s t e m , the i m m u n o r e a c t i v i t y was e l u t e d s o m e w h a t e a r l i e r than NPK and a p p e a r e d n o w at the p o s i t i o n of o x i d i z e d N P K (Fig 3b).
Discussion HPLC c h a r a c t e r i z a t i o n of N K A - L I in human CSF r e v e a l e d that m o s t of the i m m u n o r e a c t i v i t y c o e l u t e d w i t h NPK. T r y p s i n t r e a t m e n t of this
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Vol. 46, No. 23, 1990
compound yielded NKA-LI coeluting with synthetic NKA which i n d i c a t e s that the i m m u n o r e a c t i v e c o m p o u n d c o n t a i n s the N K A sequence. It has p r e v i o u s l y been r e p o r t e d that NPK i s o l a t e d from p o r c i n e b r a i n p r o d u c e N K A u p o n t r y p s i n d i g e s t i o n (i). F u r t h e r m o r e , w h e n the N K A - L I from CSF was o x i d i z e d and r e c h r o m a t o g r a p h e d the i m m u n o r e a c t i v i t y was found at the p o s i t i o n of NPK-ox, w h i c h elutes s l i g h t l y e a r l i e r than NPK. P r e v i o u s l y , NPK has b e e n r e p o r t e d to elute b e t w e e n N K A and NKB upon r e v e r s e - p h a s e HPLC (10-14). In one r e p o r t (15), however, N P K was found to elute a f t e r NKB. The o b s e r v a t i o n in the p r e s e n t study that the r e t e n t i o n time of N P K and N P K - o x (but not of the o t h e r t a c h y k i n i n s ) w e r e d i f f e r e n t u s i n g two a p p a r e n t l y i d e n t i c a l c o l u m n s u n d e r i d e n t i c a l e l u t i o n c o n d i t i o n s was s u r p r i s i n g e s p e c i a l l y since it r e s u l t e d in d i f f e r e n t e l u t i o n o r d e r s of NPK and NKB. The r e t e n t i o n of c o m p o u n d s on r e v e r s e - p h a s e c o l u m n s is h o w e v e r c o n s i d e r e d to be due to a m i x e d r e t e n t i o n m e c h a n i s m and it is thus p o s s i b l e that small c o l u m n - to c o l u m n d i f f e r e n c e s of the s t a t i o n a r y p h a s e can cause a c h a n g e of the e l u t i o n o r d e r of s u b s t a n c e s that elute r a t h e r close to each o t h e r (as e.g. N P K and N K B in the p r e s e n t study). The m a i n N K A - L I c o m p o n e n t in CSF also had d i f f e r e n t r e t e n t i o n times on the two c o l u m n s but c o e l u t e d always w i t h s y n t h e t i c NPK, also i n d i c a t i n g that the s u b s t a n c e is i n d e e d i d e n t i c a l to NPK. In a d d i t i o n to NPK, a n o t h e r N - t e r m i n a l l y e x t e n d e d form of NKA, NPY, has r e c e n t l y been i d e n t i f i e d in m a m m a l i a n t i s s u e s (2). The N K A a n t i s e r u m , N K A 5, reacts also w i t h NP7. However, this p e p t i d e e l u t e d at m u c h lower c o n c e n t r a t i o n s of a c e t o n i t r i l e than NPK and a p p e a r s c l o s e to the p o s i t i o n of NKA. We c o u l d t h e r e f o r e e x c l u d e that the m a j o r c o m p o n e n t of N K A - L I in CSF is NPy. F r o m the p r e s e n t r e s u l t s we c o n c l u d e that the c o m p o u n d r e s p o n s i b l e for the m a i n N K A - L I in CSF is m o s t l i k e l y to be NPK. The c o n c e n t r a t i o n s of N K A - L I in CSF from h e a l t h y s u b j e c t s w e r e m e a s u r e d by H P L C - R I A u s i n g N K A as standard. E x p r e s s e d in NPK e q u i v a l e n t s , o b t a i n e d by c o r r e c t i n g the r e s u l t s for c r o s s - r e a c t i v i t y and r e c o v e r y of N P K (see M a t e r i a l s and Methods) the c o n c e n t r a t i o n was c a l c u l a t e d to be 23 + ii p m o l / L (mean + SD; n=6). This transf o r m a t i o n seems r e a s o n a b l e since the d T s p l a c e m e n t c u r v e s of N K A and N P K w e r e p a r a l l e l . The H P L C - R I A m e t h o d was d e v e l o p e d for the a n a l y s i s p r i m a r i l y of N K A and we have a h i g h r e c o v e r y of this peptide. The low but r e p r o d u c i b l e r e c o v e r y of 33 + 6% t h r o u g h the e n t i r e a n a l y t i c a l p r o c e d u r e for N P K is p r o b a b l y due to a b s o r p t i o n to v a r i o u s c o m p o n e n t s of the a n a l y t i c a l system. M a c D o n a l d et al (16) have d o c u m e n t e d the p r o b l e m s w i t h N P K and a b s o r p t i o n . N P K has p r e v i o u s l y been found to be one of the m a j o r t a c h y k i n i n s in p l a s m a and in a c e t i c acid e x t r a c t s of tumor tissue from c a r c i n o i d p a t i e n t s (I0). N P K has b i o l o g i c a l a c t i v i t y in several s y s t e m s and T a t e m o t o et al (i) have s u g g e s t e d that NPK m i g h t be a n e u r o m e s s e n g e r in i t s e l f and not just a p r e c u r s o r of NKA. This h y p o t h e s i s is s t r e n g t h e n e d by the r e s u l t s of D i e z - G u e r r a et al (17) w h i c h shows that NPK can be r e l e a s e d by d e p o l a r i z i n g stimuli from rat s u b s t a n t i a nigra. It is thus p o s s i b l e that NPK in CSF m a y be used as a m a r k e r for the a c t i v i t y of c e n t r a l N P K - c o n t a i n i n g neurons.
Vol. 46, No. 23, 1990
Neuropeptide K in Human CSF
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We have r e c e n t l y d e m o n s t r a t e d that human CSF contains also an N - t e r m i n a l l y e x t e n d e d form of SP, w h i l e (undeca) SP itself could not be d e t e c t e d (7). A l s o this e x t e n d e d t a c h y k i n i n m o l e c u l e is r e l e a s e d from rat C N S - t i s s u e in vitro by d e p o l a r i z i n g stimuli s u g g e s t i n g a n e u r o m e s s e n g e r role (18). In a d d i t i o n we have o b t a i n e d data s u g g e s t i n g that SP e x t e n d e d both in the C- and N - t e r m i n a l ends is p r e s e n t in CSF (7). Thus, it seems as if the larger m o l e c u l a r forms of t a c h y k i n i n s rather than the "classical" ones are p r e s e n t in human CSF. One r e a s o n for this could be that the e x t e n d e d c o m p a r e d w i t h the smaller t a c h y k i n i n s are more m e t a b o l i c a l l y stable in the extrac e l l u l a r space or in the CSF. This subject o b v i o u s l y deserves further i n v e s t i g a t i o n . However, it has been r e p o r t e d that the p l a s m a half life of NPK is longer than that of N K A when these p e p t i d e s are infused i n t r a v e n o u s l y in guinea pigs (19). A h i g h r e s i s t a n c e of NPK to d e g r a d a t i o n w o u l d make it less likely to have a role as a transmitter, but it is also p o s s i b l e that NPK has long term effects d i f f e r e n t from a c l a s s i c a l transmitter. In p a r a l l e l to our own findings on CSF tachykinins, N y b e r g and c o l l a b o r a t o r s have r e p o r t e d that there is more of high than of low m o l e c u l a r w e i g h t o p i o i d p e p t i d e s in human CSF, p o s s i b l y due to a higher r e s i s t a n c e of the larger m o l e c u l e s to d e g r a d i n g a c t i v i t y (20,21). In c o n c l u s i o n our results d e m o n s t r a t e the p r e s e n c e of NPK in human CSF, while N K A and NKB do not seem to be present, or occur at m u c h lower c o n c e n t r a t i o n s . The p o s s i b l e u s e f u l n e s s of NPK in CSF as a m a r k e r for the a c t i v i t y in central N P K - c o n t a i n i n g neurons d e s e r v e s investigation. Acknowledgements F i n a n c i a l support was given by the S w e d i s h M e d i c a l R e s e a r c h C o u n c i l (8270, 6836), the Bank of Sweden T e r c e n t e n a r y F o u n d a t i o n (83/38), the W a l l e n b e r g F o u n d a t i o n and the K a r o l i n s k a Institute. P r o f e s s o r Hans J~rnvall, D e p a r t m e n t of C h e m i s t r y I, K a r o l i n s k a I n s t i t u t e is g r a t e f u l l y a c k n o w l e d g e d for help with the amino acid determinations.
References i. 2. 3. 4. 5. 6. 7. 8.
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