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Identification of two pro-VIP forms in a human neuroblastoma cell line
Peptides. Vol. 7, Suppl. 1, pp. 7-15, 1986. ~ Ankho International Inc. Printed in the U.S.A.
0196-9781/86 $3.00 + .00
Identification of Two Pro-VIP Forms in a Human Neuroblastoma Cell Line MICHAL
SVOBODA,* ANNE GREGOIRE,t CHIZUKO YANAIHARA,$ NOBORU YANAIHARAt A N D J E A N C H R I S T O P H E .1
*Department o f Biochemistry and Nutrition and tDepartment of Histology Medical School, Universit# Libre de Bruxelles, Brussels, B-IO00 Belgium and SLaboratory o f Bioorganic Chemistry, ~ Shizuoka College o f Pharmacy, Shizuoka 422, Japan SVOBODA, M., A. GREGOIRE, C. YANAIHARA, N. YANAIHARA AND J. CHRISTOPHE. Identification of two pro-VIPjbrms in a human neuroblastoma cell line. PEPTIDES 7: Suppl. 1, 7-15, 1986.--The immunoreactivity of VIP and PHI standards, immobilized on a nitrocellulose membrane, was first assayed with various detection procedures. For VIP, the double bridge peroxidase-antiperoxidase (PAP) method was the most sensitive procedure, giving a detection limit of 0. I-0.3 pmol per mm z with the 4 rabbit anti-VIP antisera tested. By contrast, the detection limit of immobilized PHI was 100 times higher with the 4 rabbit anti-PHI/PHM antisera tested presumably because major antigenic sites were masked in the immobilized peptide. With this information at hand, the VIP and PHI immunoreactivity of human neuroblastoma NB-OK-I cells was tested after extraction, SDS-PAGE, electrotransfer, and PAP immunodetection. Two faint immunoreactive bands corresponding to two pro-VIP forms with an Mr of, respectively, 19 kDa and 18 kDa, were detected in undifferentiated cells. These distinct bands increased progressively and markedly during differentiation in the presence of dibutyryl cyclic AMP. In addition, two intermediary VIP forms of lower Mr (11 kDa and 6 kDa) and 3 kDa VIP itself were also present after 2 days of differentiation. The 19 kDa and 18 kDa pro-VIP forms were detected with a sensitivity several times higher than that of VIP and their staining was specific for VIP epitopes. By contrast, when using 4 rabbit antiPHI/PHM antisera, we observed essentially the strong unspecific staining of a 17 kDa polypeptide. VIP immunoreactivity was also visualized by immunocytochemistry in neuroblastoma cells cultured on glass coverslips and fixed in situ. Specific VIP staining using the PAP method was present in 10 percent of the cells in the undifferentiated state; this proportion rose to 70 percent after two days in the presence of dibutyryl cyclic AMP. The preferential immunostaining of the Golgi apparatus in positive cells was reminiscent of the high sensitivity of the PAP method for pro-VIP forms immobilized on nitrocellulose. Human neuroblastoma cell
VIP precursors
Electrotransfer
V A S O A C T I V E intestinal polypeptide (VIP), a 28 amino acid peptide originally isolated from porcine upper small intestine, was later identified as a neurotransmitter (for r e v i e w see [18]). A human n e u r o b l a s t o m a cell line, originally established in Japan and now designated N B - O K - I [27], synthesizes VIP [26,27] by processing pro-VIP, a 18 k D a VIP prec u r s o r w h o s e presence was d e m o n s t r a t e d by immunoprecipitation of proteins radiolabelled in cells cultured with (asS)methionine [17]. Dibutyryl cyclic A M P induces the differentiation of these cells and increases their VIP production l 1-fold [27] by enhancing the transcription rate o f the m - R N A encoding p r e p r o - V I P [7]. W h e n considering the nucleotide s e q u e n c e of the VIP c - D N A , c o m p l e m e n t a r y to the m - R N A w h o s e primary translation produces a p r e p r o - V I P form o f 20 k D a [17], it appears that the p r e c u r s o r protein contains, in addition to VIP, another neuropeptide of 27 amino acids: Peptide Histidine-Methionine (PHM) [9]. P H M is the human equivalent o f porcine Peptide Histidine-Isoleucine (PHI) [22,23] w h o s e i m m u n o r e a c t i v i t y is generally found in cells containing VIP [1, 3, 11]. The ~Requests for reprints should be addressed to Jean Christophe.
lmmunoblot
o c c u r r e n c e o f 3 kDa P H M in n e u r o b l a s t o m a N B - O K - 1 cells has been d e m o n s t r a t e d by c h r o m a t o g r a p h y and radioimm u n o a s s a y [8] but the p r e s e n c e o f P H M i m m u n o r e a c t i v e epitopes in pro-VIP has not yet been characterized. The structure o f the human p r e c u r s o r gene [2] has shown the ~xistence on two separate e x o n s o f the VIP and P H M coding sequences. The aims o f the present w o r k were two-fold: (1) to analyze the potency of 4 rabbit anti-VIP and 4 rabbit antiP H I / P M H antisera using a solid phase assay; and (2) to compare the VIP and P H I / P H M antigenic c o n t e n t of neuroblast o m a NB-OK-1 cells before and after differentiation under dibutyryl cyclic A M P , using S D S - P A G E and i m m u n o b l o t techniques.
METHOD
Materials Anti-VIP R-501, anti-VIP R-502, a n t i - P H I ( l - 1 5 ) R-8403,
8
SVOBODA E7" AL. ABBREVIATIONS
VIP PHI PHM SDS-PAGE PAP Mr Bt._,cAMP KDa
vasoactive intestinal peptide Peptide Histidine-lsoleucine Peptide Histidine-Methionine polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate peroxidase-antiperoxidase complex molecular radius dibutyryl cyclic AMP K-dalton
anti-PHI(20--27) R-8201, anti-PHM(13-27) R-8502, and antiPHM R-8702 rabbit antisera were prepared and characterized as described in [25] and 128]. Anti-VIP AB 35 rabbit antiserum was from Cambridge Research Biochemicals (Cambridge, U.K.) and anti-VIP RPN 1582 rabbit antiserum was from Amersham Radiochemical Centre (Bucks, U.K.). The media and materials for cell culture were from Gibco Europe (Gent, Belgium). VIP and PHI were from Sigma Chemical Company (St Louis, MO). Dibutyryl cyclic AMP was from Boehringer (Mannheim/M, FRG). Sheep and goat anti-rabbit gamma globulins and 3,3'diaminobenzidine (DAB) were from Sigma Chemical Co. and the peroxidase-antiperoxisase complex (PAP) was from Dakopatts (Glostrup, Denmark). Alternatively, these three reagents were obtained as a PAP kit from Bio-Lyon (Lyon, France). Ovalbumen (egg powder) was from BDH (Poole, U.K.). Tween 20 EIA grade and the goat anti-rabbit gamma globulin-peroxidase conjugate were from Bio-Rad (Richmond, CA). The anti-rabbit biotin-streptavidin peroxidase kit was from Amersham Radiochemical Centre. The anti-rabbit immunoglobulin-colloidal gold and silver enhancement kit was from Janssen Life Sciences Products ( Beerse, Belgium). All other reagents were of analytical grade. Methods Cell culture. Neuroblastoma NB-OK-1 was derived from a cell line established in 1973 [27]. Cells were grown in 80 cm z Nunclon tissue culture flasks in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. The cells were grown at 37°C in an atmosphere of 5% CO.,-95% air at 10WA humidity. Passages were performed 1:3 every week after mild trypsinisation. The analysis of the cell content was performed 5 to 7 days after the last passage, the 2 to 3 last days of incubation being, whenever indicated, conducted in the presence of 0.5-1.0 mM dibutyryl cyclic AMP. After two cell washes with Dulbecco's phosphate buffered saline without Ca z~ and Mg 2', the cells were mechanically detached by gentle shaking then centrifuged 5 rain at 500 × g. Cell pellets were directly solubilized at 100°C in a SDS-PAGE sample buffer made of 0.125 M Tris-HCI buffer (pH 6.8) enriched with 5% SDS, 5% 2-mercaptoethanol, 20% sucrose, and 0.02% phenol red as tracking dye. Eleetrophoresis and immunodeteetion. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 80×80×0.7 mm slab gels. The total acrylamide content was 14-15% (with 5% bis-acrylamide) for separating
gels, and 5% (with 2.7% bis-acrylamide) for stacking gels. The discontinuous Laemmli buffer system was used at buffer concentrations twice as high as those mentioned in the original method 112]. Transversal electrophoresis was conducted lbr I hour at 30 V as previously described [21] in a horizontal apparatus manufactured by Bio-Lyon (Lyon, France) and equipped with an anode made of surface conductive glass and a cathode made of stainless steel. Nitrocellulose membranes (0.2/zm pore size, bright side up) from Sartorius (G6ttingen, FRG) were used as immobilizing sheet. We verified that more than 90% of a '2sI-VIP standard was immobilized on this type of membrane after transversal electrophoresis and was not washed away after overnight exposure of the filter to PBS--0.05% Tween 20. Non-specific antibody binding sites in these nitrocellulose sheets were then blocked by incubation for I hr in 4% ovalbumen (egg powder). The primary rabbit antisera tested were diluted in I% normal sheep serum (NSS) in phosphate buffered saline (PBS). The nitrocellulose sheets were incubated with 0.1 ml/cm'-' of each primary antiserum for 2 days at 4°C in an air atmosphere saturated with water vapors. The immobilized rabbit immunoglobulins were usually detected by the double bridge peroxidase-antiperoxidase PAP method as previously described [14,15] except that 0.05~ Tween 20 was added to the washing buffers. When other detection methods were used, we followed the instructions of the manufacturers. Dot immunobinding assays [6, 10, 19] were performed by spotting 1/sl portions of the antigen solutions in the center of 5×5 mm squares of a grid marked with a pencil on a nitrocellulose sheet. The nitrocellulose membrane was processed after drying as indicated for the immunoblot procedure (see above). lmmttmwytochemisto', r[o v~sualize VIP immunoreactivity, neuroblastoma cells were cultured on glass coverslips and directly fixed for 30 rain at 4°C in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The coverslips were rinsed in phosphate buffered saline (PBS) (pH 7.4) and the cells were quickly dehydrated in, successively, 70%, 90%, and 100e/~ ethanol, then 100cX 2-propanol. They were cleared in toluol and rehydrated through 10(}'~ 2-propanol, 100e/~, 9(F/~, and 70c~ ethanol, then PBS. The action of endogenous peroxidase was blocked with 0.3% hydrogen peroxide. Each fixed culture was processes according to the peroxidaseantiperoxidase (PAP) procedure of Sternberger [20]. Antibody penetration was enhanced by the preliminary dehydration-rehydration procedure [4] and further facilitated by a 30 rain incubation with 0.4% Triton X-100 in PBS. The incubation of the three anti-V1P antisera tested was performed at a 1:2000 dilution in 1% normal sheep serum in PBS for 24 hr at 4°C. All other steps were accomplished at room temperature and the buffer used was Coons veronal buffered saline. Finally, peroxidase activity was developed in a diaminobenzidine/HeOe medium. Staining specificity was controlled with a non-immune rabbit serum.
RESULTS Solid Phase Immunodete('tion qf VIP and PHI In solid phase, the limit of detection of a given antigen depends on the performance of the primary specific antiserum and on the method revealing bound immunoglobulins. For V1P the limit of detection was tested after ira-
IDENTIFICATION OF TWO PRO-VIP FORMS
LANE
9
2
VIP (pmol)
g
3
4
5 1>
g -n
F--U3
I
30 10 3
0.3 FIG. I. Comparison of four immunobinding assays for V1P offered in serial dilution. Aliquots of I txl of VIP solutions were applied as 3-4 mm 2 dots on a 5×5 mm rectangular grid drawn on a nitrocellulose membrane. Remaining nonspecific membrane antibody binding sites were blocked in 4% ovalbumen. The membrane was incubated for 2 days at 4°C with anti-V1P R-502 antiserum used at a 1/2000 dilution, then washed and cut in strips. Bound rabbit immunoglobulins were revealed as detailed in the Method section by: I: the double bridge PAP method using sheep anti-rabbit lgG (SARGG); 2: the double bridge PAP method using goat anti-rabbit lgG (GARGG): 3: the goat anti-rabbit lgG-peroxidase conjugate (GARGG-P) used at a 1/2000 dilution: 4: the same GARGG-P conjugate used at a 1/100 dilution: 5: biotinylated anti-rabbit IgG reacting with the streplavidinbiotinylated peroxidase preformed complex.
mobilizing probes of 0.3 to 30 pmol VIP on a nitrocellulose m e m b r a n e as 3-4 mm 2 dots. A f t e r a two days incubation with anti-VIP R-502 antiserum, the rabbit immunoglobulins bound to VIP were revealed by 4 p r o c e d u r e s (Fig. 1): the double bridge PAP method using two anti-rabbit immunoglobulins (lanes 1 and 2); the anti-rabbit immunoglobulinperoxidase conjugate used at two c o n c e n t r a t i o n s (lanes 3 and 4); the biotin-streptavidin system (lane 5); and the immunogold m e t h o d that gave results as p o o r as those in lane 3 (not shown). The double bridge PAP method allowed the highest sensitivity and the best signal to noise (background staining) ratio with as little as 0.1 to 0.3 pmol V I P / m m 2 giving a visible spot. In sharp contrast with these data, the double bridge P A P method was not more sensitive than the biotinstreptavidin and i m m u n o g o l d methods w h e n detecting rabbit immunoglobulins directly immobilized on the nitrocellulose sheet (data not shown).
Using the double bridge PAP method for dot assay, we c o m p a r e d the detection limits of VIP and PHI, immunobilized on a nitrocellulose m e m b r a n e , using 4 rabbit anti-VIP and 4 rabbit a n t i - P H I / P r i M antisera. The detection limit with the 4 anti-V1P antisera utilized was acceptable (at 0.1-0.3 p m o l / m m 2) while the a n t i - P H I / P H M antisera were 100 times less potent for PHI recognition (Table 1). This contrasts with the equivalent potency of these anti-VIP anti-PHI antisera in liquid phase i.e., for radioimmuoassay [25,28]. The difference in sensitivity of the anti-VIP and antiP H I / P H M antisera, shown in solid phase by the dot assay of VIP and PHI standards, was confirmed after S D S - P A G E , electrotransfer and i m m u n o d e t e c t i o n with the PAP method (Fig. 2). The detection limit for VIP was 0.5 pmol/mm of sample well width. This value was c o m p a r a b l e to the lowest amount of VIP detectable in dot assay, showing that SDS
S V O B O D A ET AL.
10
AMOUNT OF VlP AND PHI
2 pmol
6 pmoi
20 pmo I
60 pmol
LANE MARKER
VIP anti VIP
ANTISERUM
anti PHI
anti VIP
anti PHI
anti VIP
FIG. 2. Difference in sensitivity of anti-VIP R-501 and anti-PHI(l-15) R-8403 antisera as detected by immunoblotting. SDS-PAGE of mixtures of the indicated amounts of VIP and PHI was performed on 80x 80× 0.7 mm discontinuous 15% Laemmli slab gels. The sample well width was 8 mm and methyl green was used as a sample lane marker. The peptides were then electrotransferred to a nitrocellulose membrane. The membrane was cut in the middle of each sample lane and the resulting strips were incubated alternatively with anti-VIP R-501 antiserum or anti-PHI(l-15) R-8403 antiserum, used at a 1/2000 dilution. Detection was obtained by the double bridge PAP method as described in the Method section.
TABLE l LIMIT OF DETECTION OF VIP AND PHI BY 7 ANT1SERA 1N DOT IMMUNOBIND1NG ASSAYS Antiserum A-VIP R-501 A-VIP R-502 A-VIP AB 35 A-VIP RPN 1582 A-PHI (1-15) R 8403 A-PHM R 8702 A-PHM (13-27) R 8502
VIP (pmol/mm 2)
PHI (pmol/mm 2)
0.1 0.3 0.1 0.3* ----
----10 60 30
Dot immunoassays were performed as indicated in the legend of Fig. 1 using the double bridge PAP method with sheep anti-rabbit IgG. The primary antisera mentioned were used at a 1/2000 dilution. Staining specificity was tested by a 1 day preincubation of the antisera tested with 2.5 p~M of the corresponding peptide at 4°C. The detection limit was observed by naked eye. *High background staining was obtained with antiserum A-VIP RPN 1582.
d e n a t u r a t i o n did n o t a l t e r the i m m u n o r e a c t i v i t y o f VIP. By c o m p a r i s o n , n o i m m u n o r e a c t i v i t y c o u l d be d e t e c t e d for P H I , in line w i t h o u r r e s u l t s with d o t a s s a y s , a n d c o n f i r m i n g the low p o t e n c y , in solid p h a s e , o f the a n t i - P H I a n t i s e r a .
Analysis of Neuroblastoma NB-OK-I Cell Extracts by SDS-PAGE and lmmunoblot V I P and P H M i m m u n o r e a c t i v i t y w a s first e x p l o r e d in ext r a c t s f r o m n e u r o b l a s t o m a N B - O K - I cells d i f f e r e n t i a t e d in the p r e s e n c e o f d i b u t y r y l cyclic A M P (Fig. 3). A m o n g the p r o t e i n s e l e c t r o t r a n s f e r r e d o n t o the n i t r o c e l l u l o s e s u p p o r t , two m a j o r i m m u n o r e a c t i v e b a n d s o f M r 19 k D a a n d 18 k D a were r e v e a l e d w i t h all t h r e e a n t i - V I P a n t i s e r a t e s t e d (lanes 6-8), t h a t w e r e not p r e s e n t in c o n t r o l l a n e s (1 a n d 2) indicating the p r e s e n c e of V I P e p i t o p e s in t h e s e b a n d s . B a n d s t h a t w e r e also p r e s e n t u n d e r c o n t r o l c o n d i t i o n s (lanes 1 a n d 2) or m i n o r b a n d s t h a t w e r e not d e t e c t e d with all t h r e e a n t i s e r a o b v i o u s l y reflected the c r o s s - r e a c t i v i t y o f a n t i b o d i e s raised a g a i n s t p e p t i d e s o t h e r t h a n V I P forms. l m m u n o r e a c t i v i t y with a n t i - P H I a n d a n t i - P H M a n t i s e r a was o b s e r v e d in a M r 17 k D a b a n d (lanes 3 to 5) a n d in a 12 k D a b a n d ( with a n t i - P H I ( l - 1 5 ) R-8403 a n t i s e r u m , lane 3) but t h e s e b a n d s w e r e also slightly visualized u n d e r c o n t r o l conditions (lanes 1 a n d 2). In addition, they w e r e strongly s t a i n e d w i t h acidic d y e s (not s h o w n ) a n d c o r r e s p o n d e d , t h e r e f o r e , to m a j o r p r o t e i n s in the n e u r o b l a s t o m a cells. W e t h e n studied the influence o f 1 m M d i b u t y r y l cyclic A M P o n the d e v e l o p m e n t o f t h e VIP- a n d P H M i m m u n o r e a c t i v e c o n t e n t o f n e u r o b l a s t o m a cells (Fig. 4). T h e i m m u n o r e a c t i v i t y s t a i n e d w i t h a n t i - V I P a n t i s e r u m R-502 (Fig. 4, right panel) i n c r e a s e d w h e n related to the p r o t e i n load: the 18 k D a a n d 19 k D a b a n d s , very faint u n d e r c o n t r o l c o n d i t i o n s , b e c a m e well visualized after a 2 h r i n c u b a t i o n
IDENTIFICATION OF TWO PRO-VIP FORMS
11
LF~
r~
I
I
CONTROLS co 00 Z I EO [I] [3._
__J FF EL O I
o c~
P~ @d I O'3
@d O Lf~
O Ln
EL
L~ O]
EL
EL
H
N--I
N-H
H
ZIZ EL I <[
ZIS EL I <[
Z> I <[
EL
l
TWO PRO-VIP FORMS VIP
FIG. 3. lmmunoblot of an extract of differentiated neuroblastoma NB-OK-1 cells tested with 8 antisera. The cells were preincubated for 2 days in the presence of 1 mM dibutyryl cyclic AMP, solubilized, then fractionated by SDS-PAGE using a single, large sample well. After protein transfer, the nitrocellulose sheet was cut in 8 strips that were developed with 8 antisera. These primary incubations were performed with: (1) PBS-NSS (phosphate buffered saline--l% normal sheep serum, i.e., the buffer used for diluting rabbit antiserum) (lane 1); (2) a rabbit anti-ovine prolactin (o-PRL) antiserum used to control the specifity of the first rabbit antiserum reaction (lane 2); (3) three antiPHI/PHM antisera raised against the peptides conjugated with a crude protein extract from Ascaris suilla, used at a 1/5000 dilution (lanes 3 to 5); (4) three anti-VIP antisera (lanes 6 to 8): anti-VIP R-502 and anti-VIP R-501 antisera were raised against VIP adsorbed on polyvinylpyrrolidone, and commercial anti-VIP AB 35 antiserum was raised against a V1P-haemocyanin conjugate. Antisera R-501 and R-502 was used at a 1/5000 dilution. Antiserum AB 35 being less potent was used at a 1/1000 dilution. The bound rabbit lgG were then stained with the double bridge PAP method as indicated in the Method section..
with dibutyryl cyclic A M P , and were still increasing after a 2 day incubation period. At that time, i m m u n o r a c t i v e VIP forms o f lower Mr were also detected: (a) two faint bands o f a p p r o x i m a t e l y 11 kDa and 6 kDa, that r e p r e s e n t e d probably intermediary forms o f p r o - V I P processing; and (b) a stronger 3 kDa band that comigrated with a VIP standard and was likely to represent human VIP. With the best anti-PHI antiserum available ( A - P H I (i-15) R-8403.: Table 1), the o b v i o u s immunostaining o f 17 kDa and 12 kDa bands (Fig. 4, left panel) remained in direct proportion to the protein load, and did not correlate with the a m o u n t o f pro-VIP forms. The staining intensity o f the 17 kDa band r e m a i n e d also c o n s t a n t w h e n tested with antiPHM(13-27) R-8502 antiserum (not shown). The immunostaining specificity of n e u r o b l a s t o m a extracts with anti-VIP and anti-PHI antisera was tested next
(Fig. 5). The staining of 18 kDa and 19 kDa bands with antiVIP R-502 antiserum was abolished w h e n adding 2.5/zM VIP to the antiserum, indicating that these bands contained VIP epitopes. In contrast, the staining o f the 17 kDa and 12 kDa bands with anti-PHI(I-15) R-8403 antiserum was unaffected w h e n adding 2.5 ~tM P H I to this antiserum, demonstrating that the staining of these bands was unrelated to PHI epitopes.
lmmunocytochemistry of Neuroblastoma Cells N e u r o b l a s t o m a cells w e r e g r o w n directly on glass coverslips, avoiding d e t a c h m e n t and cell alteration before staining. W h e n grown in control conditions m o s t of the cells, after haematoxylin staining, appeared small, with a little dark nucleus surrounded by thin layer o f cytoplasm. Long, relatively thin and rarely b r a n c h e d p r o c e s s e s w e r e fre-
S V O B O D A ET AL.
12
A-VIP
A-PHI 1-15 INCUBATION WITH (Bt) 2CAMP
Oh
502
VIP FORMS
2h 48h Oh 2h 48h
E 17 kOa 12 kOa
kOa *==18 kOa
: +--
1 ko+ KOa 3 Kda
FIG. 4. The influence of dibutyryl cyclic AMP on the development of immunoreactive forms in neuroblastoma NB-OK-I cells, as revealed by anti-PHI(I-15) R-8403 and anti-VIP R-502 antisera. Neuroblastoma cells were incubated under control conditions or in the presence of 1 mM dibutyryl cyclic AMP (for 2 hours or 2 days), then solubilized and submitted to SDS-PAGE. The three samples were loaded in 4 mm width sample wells in the polyacrylamide gel. The loads of total protein applied were in the proportions 4:10:5 for, respectively, control, 2 hr and 2 days samples. After transfer, the nitrocellulose sheet was cut and the strips were developed with the two antisera used at a 1/5000 dilution and revealed by the double bridge PAP method as described in the Method section. The primary antisera were: anti-PHI(I-15) R-8403 (left panel) and anti-VlP R-502 (right panel).
quently encountered. Cells with 2 expansions were the most n u m e r o u s but s o m e were bigger and multipolar. Ninety percent of these cells were not labelled with anti-VIP antisera R-501, R-502 and AB 35. H o w e v e r , a minority (10%) o f cells exhibited specific staining in a limited round j u x t a - n u c l e a r area. In cultures differentiated for 2 days in the p r e s e n c e of 0.5 m M dibutyryl cyclic A M P , s o m e of the cells retained the same aspect and size as the majority of those o b s e r v e d in control conditions. H o w e v e r , the soma and nucleus were enlarged in most cells, the neurites were elongated and the nucleus contained dispersed chromatin. VIP i m m u n o r e a c tivity was present in 70% of the cell population as a c h r o m o gen precipitate in a sizable j u x t a - n u c l e a r area of the perikaryon (Fig. 6). The location o f the dark reaction product suggested the labelling of the Golgi apparatus as o b s e r v e d in peripheral neurons by J o h a n s s o n [11]. DISCUSSION
Methodological Requirements and Limitations The 100-fold difference in the detection limits of VIP and PHI immobilized onto a nitrocellulose m e m b r a n e probably
reflected the masking, by adsorption, of major epitopes in PHI but not in VIP (Figs. 1 and 2). The m e c h a n i s m of protein binding to nitrocellulose is poorly understood [5,24] but the masking of antigenic sites by nitrocellulose is likely to be more important for small peptides than for larger proteins with multiple antigenic sites. This may be why pro-VIP forms, that represent a minor i m m u n o r e a c t i v e fraction of total VIP immunoreactivity of n e u r o b l a s t o m a cells in liquid phase [27], were more strongly stained than 3 kDa VIP on immunoblot (especially with anti-VIP antiserum R-502: Fig. 3, lane 6 and Fig. 4, right panel). In these e x p e r i m e n t s 3 kDa V1P remained stacked as a sharp band during its migration on S D S - P A G E as long as we used freshly prepared gels, and was not washed away from the nitrocellulose sheet (Figs. 2 and 4). (When using older gels, the diffusion of stacking and separating gel buffers p r o v o k e d a diffuse migration of small peptides and, consequently, their poor detection on immunoblot: Fig. 5.) Anti-VIP antisera d e t e c t e d mainly large VIP forms on immunoblot (Fig. 5). By contrast, a n t i - P H I / P H M antisera strongly stained 2 peptides that were unrelated to PHM. This artifactual staining could be due to the method used for preparing the antisera: anti-VIP R-501 and R-502 antisera were
IDENTIFICATION OF TWO PRO-VIP FORMS
ANTISERUM ADDITION
13
A-PHI 1-15
A-VIP 5O2
0 ]PHI 0 JVlP
TWO PRO-VIP 17 kDa
FORMS
:L2 kDa
LANE FIG. 5. Staining specificity of neuroblastoma immunoblot by anti-PHI(I-15) R-8403 and anti-VIP R-502 primary antisera. Extracts of neuroblastoma cells incubated for 3 days in the presence of 0.5 mM dibutyryl cyclic AMP were fractionated on SDS-PAGE using a single large sample well. After protein transfer, the nitrocellulose sheet was cut in 5 mm wide strips. The nitrocellulose strips were incubated for 2 days with the following antisera: anti-PHI(I-15) R-8403 antiserum used at a 1/2000 dilution and preincubated for 1 day without (lane 1) or with (lane 2) 2.5 tzM PHI; anti-VIP R-502 antiserum used at a 1/2000 dilution and preincubated for I day without (lane 3) or with (lane 4) 2.5 tzM VIP.
prepared against VIP adsorbed on polyvinylpyrrolidone [25], anti-VIP AB 35 and RPN 1582 antisera were prepared against VIP conjugated with keyhole limpet haemocyanin (according to the manufacturer), and all anti-PHI/PHM antisera were prepared against PHI or P H M conjugated with carbodiimide to a crude protein extract ofAscaris suilla [28]. Our data suggest that the 2 neuroblastoma peptides stained with the anti-PHI/ P H M antisera shared c o m m o n antigenic sites with unknown c o m p o n e n t s of the crude Ascaris extract. This is of course not detrimental in a radioimunoassay specific for P H I / P H M based on c o m p e t i t i o n b e t w e e n labelled and unlabelled antigens but may p r o v o k e false positive staining after immunoblotting.
The Presence o f Two Pro-V1P Forms in the Neuroblastoma NB-OK-I Cell Line and the Immunocytochemical Location o f VIP Forms in the Golgi Apparatus The immunoblot analysis of a SDS extract of neuroblast o m a N B - O K - I cells, differentiated in the p r e s e n c e of dibutyryl cyclic A M P , using anti-VIP R-502 antiserum and the P A P detection method, recognized mainly two pro-VIP forms of Mr 19 kDa and 18 kDA. The m e t h o d we used was simpler and the c o n s u m p t i o n of antiserum was not higher than that required for the autoradiography of pro-VIP forms in a S D S - P A G E run of the i m m u n o p r e c i p i t a t e of a radiolabelled extract of NB-OK-1
14
SVOBODA E T A L .
FIG. 6. Immunocytochemical detection of VIP immunoreactivity in differentiated neuroblastoma NB-OK-I cells grown on a glass coverslip. The cells were cultured for 2 days in the presence of 0.5 mM dibutyryl cyclic AMP, then fixed and stained with anti-VIP R-502 antiserum as described in the Method section. In these differentiated cells, a dark immunoprecipitate was visible in most perikaria (x 1000).
cells [17]. If antiserum specificity is similar in both methods, the sensitivity of the present method is lower, however, than that of the radioactive method. In preliminary experiments (not shown), the two pro-VIP forms migrated close to each other on SDS-PAGE, when using 2.7% bisacrylamide cross-linking in polyacrylamide gel. These forms became well separated when using 5.0%, bisacrylamide cross-linking. The homogeneous electric field generated by surface conductive electrodes also helped us to transfer proteins on the nitrocellulose membrane without any loss of resolution [21]. The autoradiograms obtained by Obata et al. [17] gave much larger bands preventing the discrimination of two pro-VIP forms. Only one m-RNA encoding for VIP has been isolated from neuroblastoma NB-OK-1 cells [9]. The existence of two pro-VIP forms must be due, therefore, to postranslational alterations such as glycosylation. In general, a glycosylated peptide migrates more slowly on SDSPAGE than a nonglycosylated peptide [16]. The preproVIP sequence deduced from c-DNA contains a putative glycosylation site (Asn-Asp-Thr) on its 68-70 amino acid sequence [9]. Unfortunately, pro-VIP forms are poorly soluble in nondenaturating media so that our attempt to test this hypothesis has so far been unsuccessful. After incubation with 1 mM dibutyryl cyclic AMP for 2 days, two added immunoreactive VIP forms of lower Mr (11 kDa and 6 kDa) and a strong 3 kDa band (that comigrated with a VIP standard) were also present, indicating the full
capacity of this cell line to produce VIP, in conformity with previous data [8,27]. The immunocytochemical analysis was conducted on cells fixed in situ, i.e., remaining attached to their growing surface. The number of VIP positive cells increased from 10 percent in control conditions (i.e., in the absence of dibutyryl cyclic AMP) to 70 percent after 2 days in the presence of dibutyryl cyclic AMP, suggesting the relative homogeneity, in this respect, of the present NB-OK-I cell line. In previous studies, neuroblastoma cells were scraped and fixed in suspension, i.e., had lost their original morphology before immunocytochemical detection [26,27]: no more than 10 percent of these cells were found to be VIP-positive after 2 days of incubation with dibutyryl cyclic AMP. In the present study, the Golgi apparatus was by far the most prominent site of immunostaining in cells submitted to dibutyryl cyclic AMP, a likely location for the processing of pro-VIP form(s) (Fig. 6). ACKNOWLEDGEMENTS
Aided by Grant 5 ROI-AM 17010-9 from the National Institutes of Health (USA), Grants 3.4504.81 and 3.4530.789 from the Fonds de la Recherche Scientifique Medicale (Belgium), a grant from the Queen Elisabeth Foundation (Belgium), and by the Minist~re de l'Emploi et du Travail (Belgium). The authors are greatly indebted to Dr. F. Leroy for allowing us to use his facilities, to G. Bastin and C. Hubeau for technical assistance and to F. Tillemans for initiation to cell culture.
REFERENCES
1. Bishop, A. E., J. M. Polak, Y. Yiangou, N. D. Christofides and S. R. Bloom. The distributions of PHI and VIP in porcine gut and their co-localisation to a proportion of intrinsic ganglion cells. Peptides 5: 255-259, 1984.
2. Bodner, M., M. Fridkin and 1. Gozes. Coding sequences for vasoactive intestinal peptide and PHM-27 peptide are located on two adjacent exons in the human genome. Proc Natl Acad Sci USA 82: 3548-3551, 1985.
IDENTIFICATION OF TWO PRO-VIP FORMS
3. Christofides, N. D., J. M. Polak and S. R. Bloom. Studies on the distribution of PHI in mammals. Peptides 5: 261-266, 1984. 4. Costa, M., R. Buffa, J. B. Furness and E. Solcia. Immunohistochemical localization of polypeptides in peripheral autonomic nerves using whole mount preparations. Histochemistry 65: 157165. 1980. 5. Gershoni, J. M. and G. E. Palade. Protein blotting: principles and applications. Anal Biochem 131: 1-15, 1983. 6. Hawkes, R., E. Niday and J. Gordon. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119: 142-147, 1982. 7. Hayakawa, Y., K. Obata, N. ltoh, N. Yanaihara and H. Okamoto. Cyclic AMP regulation of pro-vasoactive intestinal polypeptide/PHM-27 synthesis in human neuroblastoma cells. J Biol Chem 259:9207-921 I, 1984. 8. Hoshino, M., C. Yanaihara, K. Ogino, K. Iguchi, H. Sato, T. Suzuki and N. Yanaihara. Production of VIP- and PHM (Human PHl)-related peptides in human neuroblastoma cells. Peptides 5: 155-160, 1984. 9. ltoh, N., K. Obata, N. Yanaihara and H. Okamoto. Human preprovasoactive intestinal polypeptide contains a novel PHI-27-1ike peptide, PHM-27. Nature 304: 547-549, 1983. 10. Jahn, R., W. Schiebler and P. Greengard. A quantitative dotimmunobinding assay for proteins using nitrocellulose membrane filters. Proc Natl Acad Sci USA 81: 1684-1687, 1984. 11. Johansson, O. Localization of vasoactive intestinal polypeptide- and avian pancreatic polypeptide-like immunoreactivity in the Golgi apparatus of peripheral neurons. Brain Res 262: 71-78, 1983. 12. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970. 13. Lundberg, J. M., J. Fahrenkrug, O. Larsson and A. ,~ngg/trd. Corelease of vasoactive intestinal polypeptide and peptide histidine isoleucine in relation to atropine-resistant vasodilation in cat submandibular salivary gland. Neurosci Lett 52: 37-42, 1984. 14. Meuris, S., M. Svoboda, M. Vilamala, J. Christophe and C. Robyn. Monomeric pituitary growth hormone and prolactin variants in man characterized by immunoperoxidase electrophoresis. FEBS Lett 154:111-115, 1983. 15. Meuris, S., M. Svoboda, J. Christophe and C. Robyn. Cleaving of disulfide bridges and apparent molecular weight of human prolactin variants as revealed by immunoperoxidase electrophoresis. Anal Biochem 143: 163-169, 1984. 16. Meuris, S., M. Svoboda, J. Christophe and C. Robyn. Evidence for a glycosylated prolactin variant in human pituitary and amniotic fluid. In: Prolactin, Basic and Clinical Correlates, vol 1, edited by R. M. MacLeod, M. O. Thorner and U. Scapagnini. Podova: Liviana Press, 1985, Fidia Research Series, pp. 488-493.
15
17. Obata, K., N. Itoh, H. Okamoto, C. Yanaihara, N. Yanaihara and T. Suzuki. Identification and processing of biosynthetic precursors to vasoactive intestinal polypeptide in human neuroblastoma cells. FEBS Lett 136: 123-126. 1981. 18. Said, S. I. Vasoactive intestinal polypeptide (VIP): Current status. Peptides 5: 143-150, 1984. 19. Smith, E., K. Roberts, G. W. Butcher and G. Galfre. Monoclonal antibody screening: two methods using antigens immobilized on nitrocellulose. Anal Biochem 138:119-124, 1984. 20. Sternberger, L. A. The unlabeled antibody peroxidaseantiperoxidase (PAP) method. In: lmmunocytochemisto,, 2nd edition, New York: John Wiley and Sons, 1979, pp. 104-169. 21. Svoboda, M., S. Meuris, C. Robyn and J. Christophe. Rapid electrotransfer of proteins from polyacrylamide gel to nitrocellulose membrane using surface conductive glass as anode. Anal Biochem 151: 16-23, 1985. 22. Tatemoto, K. and V. Mutt. Isolation and characterization of a novel intestinal peptide porcine PHI (PHI-27), a new member of the glucagon-secretin family. Proc Natl Acad Sci USA 78: 6603-6607, 1981. 23. Tatemoto, K., H. J6rnvall, T. J. MacDonald, M. Carlquist, V. L. W. Go, C. Johansson and V. Mutt. Isolation and primary structure of human PHI (peptide HI). FEBS Lett 174: 258-261, 1984. 24. Wallis, C., J. L. Melnick and C. P. Gerba. Concentration of viruses from water by membrane chromatography. Anntt Rev Microbiol 33: 413-437, 1979. 25. Yanaihara, N., M. Sakagami, H. Sato, K. Yamamoto, T. Hashimoto, C. Yanaihara, Z. lto, K. Yamaguchi and K. Abe. Immunological aspects of secretin, substance P, and V1P. Gastroenterology 72: 803-810, 1977. 26. Yanaihara, N., S. Kobayashi, H. Sato, C. Yanaihara, M. Sakagami, N. Sakura, T. Hashimoto, S. lshikawa, T. lwanaga and T. Fujita. Vasoactive intestinal polypeptide-like immunoreactivity in a human neuroblastoma cell line and the coexistence of other neuropeptide immunoreactivity in the cell line. Endocrinol Jpn 27: Suppl 1, 37-42, 1980. 27. Yanaihara, N., T. Suzuki, H. Sato, M. Hoshino, Y. Okaru and C. Yanaihara. Dibutyryl cAMP stimulation of production and release of VIP-like immunoreactivity in a human neuroblastoma cell line. Biomed Res 2: 728-734, 1981. 28. Yanaihara. N., C. Yanaihara, K. Nokihara, K. Iguchi, S. Fukata, M. Tanaka, Y. Yamamoto and T. Mochizuki. lmmunochemical study on PHI/PHM with use of synthetic peptides. Peptides 5: 247-254, 1984.
0196-9781/86 $3.00 + .00
Identification of Two Pro-VIP Forms in a Human Neuroblastoma Cell Line MICHAL
SVOBODA,* ANNE GREGOIRE,t CHIZUKO YANAIHARA,$ NOBORU YANAIHARAt A N D J E A N C H R I S T O P H E .1
*Department o f Biochemistry and Nutrition and tDepartment of Histology Medical School, Universit# Libre de Bruxelles, Brussels, B-IO00 Belgium and SLaboratory o f Bioorganic Chemistry, ~ Shizuoka College o f Pharmacy, Shizuoka 422, Japan SVOBODA, M., A. GREGOIRE, C. YANAIHARA, N. YANAIHARA AND J. CHRISTOPHE. Identification of two pro-VIPjbrms in a human neuroblastoma cell line. PEPTIDES 7: Suppl. 1, 7-15, 1986.--The immunoreactivity of VIP and PHI standards, immobilized on a nitrocellulose membrane, was first assayed with various detection procedures. For VIP, the double bridge peroxidase-antiperoxidase (PAP) method was the most sensitive procedure, giving a detection limit of 0. I-0.3 pmol per mm z with the 4 rabbit anti-VIP antisera tested. By contrast, the detection limit of immobilized PHI was 100 times higher with the 4 rabbit anti-PHI/PHM antisera tested presumably because major antigenic sites were masked in the immobilized peptide. With this information at hand, the VIP and PHI immunoreactivity of human neuroblastoma NB-OK-I cells was tested after extraction, SDS-PAGE, electrotransfer, and PAP immunodetection. Two faint immunoreactive bands corresponding to two pro-VIP forms with an Mr of, respectively, 19 kDa and 18 kDa, were detected in undifferentiated cells. These distinct bands increased progressively and markedly during differentiation in the presence of dibutyryl cyclic AMP. In addition, two intermediary VIP forms of lower Mr (11 kDa and 6 kDa) and 3 kDa VIP itself were also present after 2 days of differentiation. The 19 kDa and 18 kDa pro-VIP forms were detected with a sensitivity several times higher than that of VIP and their staining was specific for VIP epitopes. By contrast, when using 4 rabbit antiPHI/PHM antisera, we observed essentially the strong unspecific staining of a 17 kDa polypeptide. VIP immunoreactivity was also visualized by immunocytochemistry in neuroblastoma cells cultured on glass coverslips and fixed in situ. Specific VIP staining using the PAP method was present in 10 percent of the cells in the undifferentiated state; this proportion rose to 70 percent after two days in the presence of dibutyryl cyclic AMP. The preferential immunostaining of the Golgi apparatus in positive cells was reminiscent of the high sensitivity of the PAP method for pro-VIP forms immobilized on nitrocellulose. Human neuroblastoma cell
VIP precursors
Electrotransfer
V A S O A C T I V E intestinal polypeptide (VIP), a 28 amino acid peptide originally isolated from porcine upper small intestine, was later identified as a neurotransmitter (for r e v i e w see [18]). A human n e u r o b l a s t o m a cell line, originally established in Japan and now designated N B - O K - I [27], synthesizes VIP [26,27] by processing pro-VIP, a 18 k D a VIP prec u r s o r w h o s e presence was d e m o n s t r a t e d by immunoprecipitation of proteins radiolabelled in cells cultured with (asS)methionine [17]. Dibutyryl cyclic A M P induces the differentiation of these cells and increases their VIP production l 1-fold [27] by enhancing the transcription rate o f the m - R N A encoding p r e p r o - V I P [7]. W h e n considering the nucleotide s e q u e n c e of the VIP c - D N A , c o m p l e m e n t a r y to the m - R N A w h o s e primary translation produces a p r e p r o - V I P form o f 20 k D a [17], it appears that the p r e c u r s o r protein contains, in addition to VIP, another neuropeptide of 27 amino acids: Peptide Histidine-Methionine (PHM) [9]. P H M is the human equivalent o f porcine Peptide Histidine-Isoleucine (PHI) [22,23] w h o s e i m m u n o r e a c t i v i t y is generally found in cells containing VIP [1, 3, 11]. The ~Requests for reprints should be addressed to Jean Christophe.
lmmunoblot
o c c u r r e n c e o f 3 kDa P H M in n e u r o b l a s t o m a N B - O K - 1 cells has been d e m o n s t r a t e d by c h r o m a t o g r a p h y and radioimm u n o a s s a y [8] but the p r e s e n c e o f P H M i m m u n o r e a c t i v e epitopes in pro-VIP has not yet been characterized. The structure o f the human p r e c u r s o r gene [2] has shown the ~xistence on two separate e x o n s o f the VIP and P H M coding sequences. The aims o f the present w o r k were two-fold: (1) to analyze the potency of 4 rabbit anti-VIP and 4 rabbit antiP H I / P M H antisera using a solid phase assay; and (2) to compare the VIP and P H I / P H M antigenic c o n t e n t of neuroblast o m a NB-OK-1 cells before and after differentiation under dibutyryl cyclic A M P , using S D S - P A G E and i m m u n o b l o t techniques.
METHOD
Materials Anti-VIP R-501, anti-VIP R-502, a n t i - P H I ( l - 1 5 ) R-8403,
8
SVOBODA E7" AL. ABBREVIATIONS
VIP PHI PHM SDS-PAGE PAP Mr Bt._,cAMP KDa
vasoactive intestinal peptide Peptide Histidine-lsoleucine Peptide Histidine-Methionine polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate peroxidase-antiperoxidase complex molecular radius dibutyryl cyclic AMP K-dalton
anti-PHI(20--27) R-8201, anti-PHM(13-27) R-8502, and antiPHM R-8702 rabbit antisera were prepared and characterized as described in [25] and 128]. Anti-VIP AB 35 rabbit antiserum was from Cambridge Research Biochemicals (Cambridge, U.K.) and anti-VIP RPN 1582 rabbit antiserum was from Amersham Radiochemical Centre (Bucks, U.K.). The media and materials for cell culture were from Gibco Europe (Gent, Belgium). VIP and PHI were from Sigma Chemical Company (St Louis, MO). Dibutyryl cyclic AMP was from Boehringer (Mannheim/M, FRG). Sheep and goat anti-rabbit gamma globulins and 3,3'diaminobenzidine (DAB) were from Sigma Chemical Co. and the peroxidase-antiperoxisase complex (PAP) was from Dakopatts (Glostrup, Denmark). Alternatively, these three reagents were obtained as a PAP kit from Bio-Lyon (Lyon, France). Ovalbumen (egg powder) was from BDH (Poole, U.K.). Tween 20 EIA grade and the goat anti-rabbit gamma globulin-peroxidase conjugate were from Bio-Rad (Richmond, CA). The anti-rabbit biotin-streptavidin peroxidase kit was from Amersham Radiochemical Centre. The anti-rabbit immunoglobulin-colloidal gold and silver enhancement kit was from Janssen Life Sciences Products ( Beerse, Belgium). All other reagents were of analytical grade. Methods Cell culture. Neuroblastoma NB-OK-1 was derived from a cell line established in 1973 [27]. Cells were grown in 80 cm z Nunclon tissue culture flasks in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. The cells were grown at 37°C in an atmosphere of 5% CO.,-95% air at 10WA humidity. Passages were performed 1:3 every week after mild trypsinisation. The analysis of the cell content was performed 5 to 7 days after the last passage, the 2 to 3 last days of incubation being, whenever indicated, conducted in the presence of 0.5-1.0 mM dibutyryl cyclic AMP. After two cell washes with Dulbecco's phosphate buffered saline without Ca z~ and Mg 2', the cells were mechanically detached by gentle shaking then centrifuged 5 rain at 500 × g. Cell pellets were directly solubilized at 100°C in a SDS-PAGE sample buffer made of 0.125 M Tris-HCI buffer (pH 6.8) enriched with 5% SDS, 5% 2-mercaptoethanol, 20% sucrose, and 0.02% phenol red as tracking dye. Eleetrophoresis and immunodeteetion. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 80×80×0.7 mm slab gels. The total acrylamide content was 14-15% (with 5% bis-acrylamide) for separating
gels, and 5% (with 2.7% bis-acrylamide) for stacking gels. The discontinuous Laemmli buffer system was used at buffer concentrations twice as high as those mentioned in the original method 112]. Transversal electrophoresis was conducted lbr I hour at 30 V as previously described [21] in a horizontal apparatus manufactured by Bio-Lyon (Lyon, France) and equipped with an anode made of surface conductive glass and a cathode made of stainless steel. Nitrocellulose membranes (0.2/zm pore size, bright side up) from Sartorius (G6ttingen, FRG) were used as immobilizing sheet. We verified that more than 90% of a '2sI-VIP standard was immobilized on this type of membrane after transversal electrophoresis and was not washed away after overnight exposure of the filter to PBS--0.05% Tween 20. Non-specific antibody binding sites in these nitrocellulose sheets were then blocked by incubation for I hr in 4% ovalbumen (egg powder). The primary rabbit antisera tested were diluted in I% normal sheep serum (NSS) in phosphate buffered saline (PBS). The nitrocellulose sheets were incubated with 0.1 ml/cm'-' of each primary antiserum for 2 days at 4°C in an air atmosphere saturated with water vapors. The immobilized rabbit immunoglobulins were usually detected by the double bridge peroxidase-antiperoxidase PAP method as previously described [14,15] except that 0.05~ Tween 20 was added to the washing buffers. When other detection methods were used, we followed the instructions of the manufacturers. Dot immunobinding assays [6, 10, 19] were performed by spotting 1/sl portions of the antigen solutions in the center of 5×5 mm squares of a grid marked with a pencil on a nitrocellulose sheet. The nitrocellulose membrane was processed after drying as indicated for the immunoblot procedure (see above). lmmttmwytochemisto', r[o v~sualize VIP immunoreactivity, neuroblastoma cells were cultured on glass coverslips and directly fixed for 30 rain at 4°C in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The coverslips were rinsed in phosphate buffered saline (PBS) (pH 7.4) and the cells were quickly dehydrated in, successively, 70%, 90%, and 100e/~ ethanol, then 100cX 2-propanol. They were cleared in toluol and rehydrated through 10(}'~ 2-propanol, 100e/~, 9(F/~, and 70c~ ethanol, then PBS. The action of endogenous peroxidase was blocked with 0.3% hydrogen peroxide. Each fixed culture was processes according to the peroxidaseantiperoxidase (PAP) procedure of Sternberger [20]. Antibody penetration was enhanced by the preliminary dehydration-rehydration procedure [4] and further facilitated by a 30 rain incubation with 0.4% Triton X-100 in PBS. The incubation of the three anti-V1P antisera tested was performed at a 1:2000 dilution in 1% normal sheep serum in PBS for 24 hr at 4°C. All other steps were accomplished at room temperature and the buffer used was Coons veronal buffered saline. Finally, peroxidase activity was developed in a diaminobenzidine/HeOe medium. Staining specificity was controlled with a non-immune rabbit serum.
RESULTS Solid Phase Immunodete('tion qf VIP and PHI In solid phase, the limit of detection of a given antigen depends on the performance of the primary specific antiserum and on the method revealing bound immunoglobulins. For V1P the limit of detection was tested after ira-
IDENTIFICATION OF TWO PRO-VIP FORMS
LANE
9
2
VIP (pmol)
g
3
4
5 1>
g -n
F--U3
I
30 10 3
0.3 FIG. I. Comparison of four immunobinding assays for V1P offered in serial dilution. Aliquots of I txl of VIP solutions were applied as 3-4 mm 2 dots on a 5×5 mm rectangular grid drawn on a nitrocellulose membrane. Remaining nonspecific membrane antibody binding sites were blocked in 4% ovalbumen. The membrane was incubated for 2 days at 4°C with anti-V1P R-502 antiserum used at a 1/2000 dilution, then washed and cut in strips. Bound rabbit immunoglobulins were revealed as detailed in the Method section by: I: the double bridge PAP method using sheep anti-rabbit lgG (SARGG); 2: the double bridge PAP method using goat anti-rabbit lgG (GARGG): 3: the goat anti-rabbit lgG-peroxidase conjugate (GARGG-P) used at a 1/2000 dilution: 4: the same GARGG-P conjugate used at a 1/100 dilution: 5: biotinylated anti-rabbit IgG reacting with the streplavidinbiotinylated peroxidase preformed complex.
mobilizing probes of 0.3 to 30 pmol VIP on a nitrocellulose m e m b r a n e as 3-4 mm 2 dots. A f t e r a two days incubation with anti-VIP R-502 antiserum, the rabbit immunoglobulins bound to VIP were revealed by 4 p r o c e d u r e s (Fig. 1): the double bridge PAP method using two anti-rabbit immunoglobulins (lanes 1 and 2); the anti-rabbit immunoglobulinperoxidase conjugate used at two c o n c e n t r a t i o n s (lanes 3 and 4); the biotin-streptavidin system (lane 5); and the immunogold m e t h o d that gave results as p o o r as those in lane 3 (not shown). The double bridge PAP method allowed the highest sensitivity and the best signal to noise (background staining) ratio with as little as 0.1 to 0.3 pmol V I P / m m 2 giving a visible spot. In sharp contrast with these data, the double bridge P A P method was not more sensitive than the biotinstreptavidin and i m m u n o g o l d methods w h e n detecting rabbit immunoglobulins directly immobilized on the nitrocellulose sheet (data not shown).
Using the double bridge PAP method for dot assay, we c o m p a r e d the detection limits of VIP and PHI, immunobilized on a nitrocellulose m e m b r a n e , using 4 rabbit anti-VIP and 4 rabbit a n t i - P H I / P r i M antisera. The detection limit with the 4 anti-V1P antisera utilized was acceptable (at 0.1-0.3 p m o l / m m 2) while the a n t i - P H I / P H M antisera were 100 times less potent for PHI recognition (Table 1). This contrasts with the equivalent potency of these anti-VIP anti-PHI antisera in liquid phase i.e., for radioimmuoassay [25,28]. The difference in sensitivity of the anti-VIP and antiP H I / P H M antisera, shown in solid phase by the dot assay of VIP and PHI standards, was confirmed after S D S - P A G E , electrotransfer and i m m u n o d e t e c t i o n with the PAP method (Fig. 2). The detection limit for VIP was 0.5 pmol/mm of sample well width. This value was c o m p a r a b l e to the lowest amount of VIP detectable in dot assay, showing that SDS
S V O B O D A ET AL.
10
AMOUNT OF VlP AND PHI
2 pmol
6 pmoi
20 pmo I
60 pmol
LANE MARKER
VIP anti VIP
ANTISERUM
anti PHI
anti VIP
anti PHI
anti VIP
FIG. 2. Difference in sensitivity of anti-VIP R-501 and anti-PHI(l-15) R-8403 antisera as detected by immunoblotting. SDS-PAGE of mixtures of the indicated amounts of VIP and PHI was performed on 80x 80× 0.7 mm discontinuous 15% Laemmli slab gels. The sample well width was 8 mm and methyl green was used as a sample lane marker. The peptides were then electrotransferred to a nitrocellulose membrane. The membrane was cut in the middle of each sample lane and the resulting strips were incubated alternatively with anti-VIP R-501 antiserum or anti-PHI(l-15) R-8403 antiserum, used at a 1/2000 dilution. Detection was obtained by the double bridge PAP method as described in the Method section.
TABLE l LIMIT OF DETECTION OF VIP AND PHI BY 7 ANT1SERA 1N DOT IMMUNOBIND1NG ASSAYS Antiserum A-VIP R-501 A-VIP R-502 A-VIP AB 35 A-VIP RPN 1582 A-PHI (1-15) R 8403 A-PHM R 8702 A-PHM (13-27) R 8502
VIP (pmol/mm 2)
PHI (pmol/mm 2)
0.1 0.3 0.1 0.3* ----
----10 60 30
Dot immunoassays were performed as indicated in the legend of Fig. 1 using the double bridge PAP method with sheep anti-rabbit IgG. The primary antisera mentioned were used at a 1/2000 dilution. Staining specificity was tested by a 1 day preincubation of the antisera tested with 2.5 p~M of the corresponding peptide at 4°C. The detection limit was observed by naked eye. *High background staining was obtained with antiserum A-VIP RPN 1582.
d e n a t u r a t i o n did n o t a l t e r the i m m u n o r e a c t i v i t y o f VIP. By c o m p a r i s o n , n o i m m u n o r e a c t i v i t y c o u l d be d e t e c t e d for P H I , in line w i t h o u r r e s u l t s with d o t a s s a y s , a n d c o n f i r m i n g the low p o t e n c y , in solid p h a s e , o f the a n t i - P H I a n t i s e r a .
Analysis of Neuroblastoma NB-OK-I Cell Extracts by SDS-PAGE and lmmunoblot V I P and P H M i m m u n o r e a c t i v i t y w a s first e x p l o r e d in ext r a c t s f r o m n e u r o b l a s t o m a N B - O K - I cells d i f f e r e n t i a t e d in the p r e s e n c e o f d i b u t y r y l cyclic A M P (Fig. 3). A m o n g the p r o t e i n s e l e c t r o t r a n s f e r r e d o n t o the n i t r o c e l l u l o s e s u p p o r t , two m a j o r i m m u n o r e a c t i v e b a n d s o f M r 19 k D a a n d 18 k D a were r e v e a l e d w i t h all t h r e e a n t i - V I P a n t i s e r a t e s t e d (lanes 6-8), t h a t w e r e not p r e s e n t in c o n t r o l l a n e s (1 a n d 2) indicating the p r e s e n c e of V I P e p i t o p e s in t h e s e b a n d s . B a n d s t h a t w e r e also p r e s e n t u n d e r c o n t r o l c o n d i t i o n s (lanes 1 a n d 2) or m i n o r b a n d s t h a t w e r e not d e t e c t e d with all t h r e e a n t i s e r a o b v i o u s l y reflected the c r o s s - r e a c t i v i t y o f a n t i b o d i e s raised a g a i n s t p e p t i d e s o t h e r t h a n V I P forms. l m m u n o r e a c t i v i t y with a n t i - P H I a n d a n t i - P H M a n t i s e r a was o b s e r v e d in a M r 17 k D a b a n d (lanes 3 to 5) a n d in a 12 k D a b a n d ( with a n t i - P H I ( l - 1 5 ) R-8403 a n t i s e r u m , lane 3) but t h e s e b a n d s w e r e also slightly visualized u n d e r c o n t r o l conditions (lanes 1 a n d 2). In addition, they w e r e strongly s t a i n e d w i t h acidic d y e s (not s h o w n ) a n d c o r r e s p o n d e d , t h e r e f o r e , to m a j o r p r o t e i n s in the n e u r o b l a s t o m a cells. W e t h e n studied the influence o f 1 m M d i b u t y r y l cyclic A M P o n the d e v e l o p m e n t o f t h e VIP- a n d P H M i m m u n o r e a c t i v e c o n t e n t o f n e u r o b l a s t o m a cells (Fig. 4). T h e i m m u n o r e a c t i v i t y s t a i n e d w i t h a n t i - V I P a n t i s e r u m R-502 (Fig. 4, right panel) i n c r e a s e d w h e n related to the p r o t e i n load: the 18 k D a a n d 19 k D a b a n d s , very faint u n d e r c o n t r o l c o n d i t i o n s , b e c a m e well visualized after a 2 h r i n c u b a t i o n
IDENTIFICATION OF TWO PRO-VIP FORMS
11
LF~
r~
I
I
CONTROLS co 00 Z I EO [I] [3._
__J FF EL O I
o c~
P~ @d I O'3
@d O Lf~
O Ln
EL
L~ O]
EL
EL
H
N--I
N-H
H
ZIZ EL I <[
ZIS EL I <[
Z> I <[
EL
l
TWO PRO-VIP FORMS VIP
FIG. 3. lmmunoblot of an extract of differentiated neuroblastoma NB-OK-1 cells tested with 8 antisera. The cells were preincubated for 2 days in the presence of 1 mM dibutyryl cyclic AMP, solubilized, then fractionated by SDS-PAGE using a single, large sample well. After protein transfer, the nitrocellulose sheet was cut in 8 strips that were developed with 8 antisera. These primary incubations were performed with: (1) PBS-NSS (phosphate buffered saline--l% normal sheep serum, i.e., the buffer used for diluting rabbit antiserum) (lane 1); (2) a rabbit anti-ovine prolactin (o-PRL) antiserum used to control the specifity of the first rabbit antiserum reaction (lane 2); (3) three antiPHI/PHM antisera raised against the peptides conjugated with a crude protein extract from Ascaris suilla, used at a 1/5000 dilution (lanes 3 to 5); (4) three anti-VIP antisera (lanes 6 to 8): anti-VIP R-502 and anti-VIP R-501 antisera were raised against VIP adsorbed on polyvinylpyrrolidone, and commercial anti-VIP AB 35 antiserum was raised against a V1P-haemocyanin conjugate. Antisera R-501 and R-502 was used at a 1/5000 dilution. Antiserum AB 35 being less potent was used at a 1/1000 dilution. The bound rabbit lgG were then stained with the double bridge PAP method as indicated in the Method section..
with dibutyryl cyclic A M P , and were still increasing after a 2 day incubation period. At that time, i m m u n o r a c t i v e VIP forms o f lower Mr were also detected: (a) two faint bands o f a p p r o x i m a t e l y 11 kDa and 6 kDa, that r e p r e s e n t e d probably intermediary forms o f p r o - V I P processing; and (b) a stronger 3 kDa band that comigrated with a VIP standard and was likely to represent human VIP. With the best anti-PHI antiserum available ( A - P H I (i-15) R-8403.: Table 1), the o b v i o u s immunostaining o f 17 kDa and 12 kDa bands (Fig. 4, left panel) remained in direct proportion to the protein load, and did not correlate with the a m o u n t o f pro-VIP forms. The staining intensity o f the 17 kDa band r e m a i n e d also c o n s t a n t w h e n tested with antiPHM(13-27) R-8502 antiserum (not shown). The immunostaining specificity of n e u r o b l a s t o m a extracts with anti-VIP and anti-PHI antisera was tested next
(Fig. 5). The staining of 18 kDa and 19 kDa bands with antiVIP R-502 antiserum was abolished w h e n adding 2.5/zM VIP to the antiserum, indicating that these bands contained VIP epitopes. In contrast, the staining o f the 17 kDa and 12 kDa bands with anti-PHI(I-15) R-8403 antiserum was unaffected w h e n adding 2.5 ~tM P H I to this antiserum, demonstrating that the staining of these bands was unrelated to PHI epitopes.
lmmunocytochemistry of Neuroblastoma Cells N e u r o b l a s t o m a cells w e r e g r o w n directly on glass coverslips, avoiding d e t a c h m e n t and cell alteration before staining. W h e n grown in control conditions m o s t of the cells, after haematoxylin staining, appeared small, with a little dark nucleus surrounded by thin layer o f cytoplasm. Long, relatively thin and rarely b r a n c h e d p r o c e s s e s w e r e fre-
S V O B O D A ET AL.
12
A-VIP
A-PHI 1-15 INCUBATION WITH (Bt) 2CAMP
Oh
502
VIP FORMS
2h 48h Oh 2h 48h
E 17 kOa 12 kOa
kOa *==18 kOa
: +--
1 ko+ KOa 3 Kda
FIG. 4. The influence of dibutyryl cyclic AMP on the development of immunoreactive forms in neuroblastoma NB-OK-I cells, as revealed by anti-PHI(I-15) R-8403 and anti-VIP R-502 antisera. Neuroblastoma cells were incubated under control conditions or in the presence of 1 mM dibutyryl cyclic AMP (for 2 hours or 2 days), then solubilized and submitted to SDS-PAGE. The three samples were loaded in 4 mm width sample wells in the polyacrylamide gel. The loads of total protein applied were in the proportions 4:10:5 for, respectively, control, 2 hr and 2 days samples. After transfer, the nitrocellulose sheet was cut and the strips were developed with the two antisera used at a 1/5000 dilution and revealed by the double bridge PAP method as described in the Method section. The primary antisera were: anti-PHI(I-15) R-8403 (left panel) and anti-VlP R-502 (right panel).
quently encountered. Cells with 2 expansions were the most n u m e r o u s but s o m e were bigger and multipolar. Ninety percent of these cells were not labelled with anti-VIP antisera R-501, R-502 and AB 35. H o w e v e r , a minority (10%) o f cells exhibited specific staining in a limited round j u x t a - n u c l e a r area. In cultures differentiated for 2 days in the p r e s e n c e of 0.5 m M dibutyryl cyclic A M P , s o m e of the cells retained the same aspect and size as the majority of those o b s e r v e d in control conditions. H o w e v e r , the soma and nucleus were enlarged in most cells, the neurites were elongated and the nucleus contained dispersed chromatin. VIP i m m u n o r e a c tivity was present in 70% of the cell population as a c h r o m o gen precipitate in a sizable j u x t a - n u c l e a r area of the perikaryon (Fig. 6). The location o f the dark reaction product suggested the labelling of the Golgi apparatus as o b s e r v e d in peripheral neurons by J o h a n s s o n [11]. DISCUSSION
Methodological Requirements and Limitations The 100-fold difference in the detection limits of VIP and PHI immobilized onto a nitrocellulose m e m b r a n e probably
reflected the masking, by adsorption, of major epitopes in PHI but not in VIP (Figs. 1 and 2). The m e c h a n i s m of protein binding to nitrocellulose is poorly understood [5,24] but the masking of antigenic sites by nitrocellulose is likely to be more important for small peptides than for larger proteins with multiple antigenic sites. This may be why pro-VIP forms, that represent a minor i m m u n o r e a c t i v e fraction of total VIP immunoreactivity of n e u r o b l a s t o m a cells in liquid phase [27], were more strongly stained than 3 kDa VIP on immunoblot (especially with anti-VIP antiserum R-502: Fig. 3, lane 6 and Fig. 4, right panel). In these e x p e r i m e n t s 3 kDa V1P remained stacked as a sharp band during its migration on S D S - P A G E as long as we used freshly prepared gels, and was not washed away from the nitrocellulose sheet (Figs. 2 and 4). (When using older gels, the diffusion of stacking and separating gel buffers p r o v o k e d a diffuse migration of small peptides and, consequently, their poor detection on immunoblot: Fig. 5.) Anti-VIP antisera d e t e c t e d mainly large VIP forms on immunoblot (Fig. 5). By contrast, a n t i - P H I / P H M antisera strongly stained 2 peptides that were unrelated to PHM. This artifactual staining could be due to the method used for preparing the antisera: anti-VIP R-501 and R-502 antisera were
IDENTIFICATION OF TWO PRO-VIP FORMS
ANTISERUM ADDITION
13
A-PHI 1-15
A-VIP 5O2
0 ]PHI 0 JVlP
TWO PRO-VIP 17 kDa
FORMS
:L2 kDa
LANE FIG. 5. Staining specificity of neuroblastoma immunoblot by anti-PHI(I-15) R-8403 and anti-VIP R-502 primary antisera. Extracts of neuroblastoma cells incubated for 3 days in the presence of 0.5 mM dibutyryl cyclic AMP were fractionated on SDS-PAGE using a single large sample well. After protein transfer, the nitrocellulose sheet was cut in 5 mm wide strips. The nitrocellulose strips were incubated for 2 days with the following antisera: anti-PHI(I-15) R-8403 antiserum used at a 1/2000 dilution and preincubated for 1 day without (lane 1) or with (lane 2) 2.5 tzM PHI; anti-VIP R-502 antiserum used at a 1/2000 dilution and preincubated for I day without (lane 3) or with (lane 4) 2.5 tzM VIP.
prepared against VIP adsorbed on polyvinylpyrrolidone [25], anti-VIP AB 35 and RPN 1582 antisera were prepared against VIP conjugated with keyhole limpet haemocyanin (according to the manufacturer), and all anti-PHI/PHM antisera were prepared against PHI or P H M conjugated with carbodiimide to a crude protein extract ofAscaris suilla [28]. Our data suggest that the 2 neuroblastoma peptides stained with the anti-PHI/ P H M antisera shared c o m m o n antigenic sites with unknown c o m p o n e n t s of the crude Ascaris extract. This is of course not detrimental in a radioimunoassay specific for P H I / P H M based on c o m p e t i t i o n b e t w e e n labelled and unlabelled antigens but may p r o v o k e false positive staining after immunoblotting.
The Presence o f Two Pro-V1P Forms in the Neuroblastoma NB-OK-I Cell Line and the Immunocytochemical Location o f VIP Forms in the Golgi Apparatus The immunoblot analysis of a SDS extract of neuroblast o m a N B - O K - I cells, differentiated in the p r e s e n c e of dibutyryl cyclic A M P , using anti-VIP R-502 antiserum and the P A P detection method, recognized mainly two pro-VIP forms of Mr 19 kDa and 18 kDA. The m e t h o d we used was simpler and the c o n s u m p t i o n of antiserum was not higher than that required for the autoradiography of pro-VIP forms in a S D S - P A G E run of the i m m u n o p r e c i p i t a t e of a radiolabelled extract of NB-OK-1
14
SVOBODA E T A L .
FIG. 6. Immunocytochemical detection of VIP immunoreactivity in differentiated neuroblastoma NB-OK-I cells grown on a glass coverslip. The cells were cultured for 2 days in the presence of 0.5 mM dibutyryl cyclic AMP, then fixed and stained with anti-VIP R-502 antiserum as described in the Method section. In these differentiated cells, a dark immunoprecipitate was visible in most perikaria (x 1000).
cells [17]. If antiserum specificity is similar in both methods, the sensitivity of the present method is lower, however, than that of the radioactive method. In preliminary experiments (not shown), the two pro-VIP forms migrated close to each other on SDS-PAGE, when using 2.7% bisacrylamide cross-linking in polyacrylamide gel. These forms became well separated when using 5.0%, bisacrylamide cross-linking. The homogeneous electric field generated by surface conductive electrodes also helped us to transfer proteins on the nitrocellulose membrane without any loss of resolution [21]. The autoradiograms obtained by Obata et al. [17] gave much larger bands preventing the discrimination of two pro-VIP forms. Only one m-RNA encoding for VIP has been isolated from neuroblastoma NB-OK-1 cells [9]. The existence of two pro-VIP forms must be due, therefore, to postranslational alterations such as glycosylation. In general, a glycosylated peptide migrates more slowly on SDSPAGE than a nonglycosylated peptide [16]. The preproVIP sequence deduced from c-DNA contains a putative glycosylation site (Asn-Asp-Thr) on its 68-70 amino acid sequence [9]. Unfortunately, pro-VIP forms are poorly soluble in nondenaturating media so that our attempt to test this hypothesis has so far been unsuccessful. After incubation with 1 mM dibutyryl cyclic AMP for 2 days, two added immunoreactive VIP forms of lower Mr (11 kDa and 6 kDa) and a strong 3 kDa band (that comigrated with a VIP standard) were also present, indicating the full
capacity of this cell line to produce VIP, in conformity with previous data [8,27]. The immunocytochemical analysis was conducted on cells fixed in situ, i.e., remaining attached to their growing surface. The number of VIP positive cells increased from 10 percent in control conditions (i.e., in the absence of dibutyryl cyclic AMP) to 70 percent after 2 days in the presence of dibutyryl cyclic AMP, suggesting the relative homogeneity, in this respect, of the present NB-OK-I cell line. In previous studies, neuroblastoma cells were scraped and fixed in suspension, i.e., had lost their original morphology before immunocytochemical detection [26,27]: no more than 10 percent of these cells were found to be VIP-positive after 2 days of incubation with dibutyryl cyclic AMP. In the present study, the Golgi apparatus was by far the most prominent site of immunostaining in cells submitted to dibutyryl cyclic AMP, a likely location for the processing of pro-VIP form(s) (Fig. 6). ACKNOWLEDGEMENTS
Aided by Grant 5 ROI-AM 17010-9 from the National Institutes of Health (USA), Grants 3.4504.81 and 3.4530.789 from the Fonds de la Recherche Scientifique Medicale (Belgium), a grant from the Queen Elisabeth Foundation (Belgium), and by the Minist~re de l'Emploi et du Travail (Belgium). The authors are greatly indebted to Dr. F. Leroy for allowing us to use his facilities, to G. Bastin and C. Hubeau for technical assistance and to F. Tillemans for initiation to cell culture.
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IDENTIFICATION OF TWO PRO-VIP FORMS
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15
17. Obata, K., N. Itoh, H. Okamoto, C. Yanaihara, N. Yanaihara and T. Suzuki. Identification and processing of biosynthetic precursors to vasoactive intestinal polypeptide in human neuroblastoma cells. FEBS Lett 136: 123-126. 1981. 18. Said, S. I. Vasoactive intestinal polypeptide (VIP): Current status. Peptides 5: 143-150, 1984. 19. Smith, E., K. Roberts, G. W. Butcher and G. Galfre. Monoclonal antibody screening: two methods using antigens immobilized on nitrocellulose. Anal Biochem 138:119-124, 1984. 20. Sternberger, L. A. The unlabeled antibody peroxidaseantiperoxidase (PAP) method. In: lmmunocytochemisto,, 2nd edition, New York: John Wiley and Sons, 1979, pp. 104-169. 21. Svoboda, M., S. Meuris, C. Robyn and J. Christophe. Rapid electrotransfer of proteins from polyacrylamide gel to nitrocellulose membrane using surface conductive glass as anode. Anal Biochem 151: 16-23, 1985. 22. Tatemoto, K. and V. Mutt. Isolation and characterization of a novel intestinal peptide porcine PHI (PHI-27), a new member of the glucagon-secretin family. Proc Natl Acad Sci USA 78: 6603-6607, 1981. 23. Tatemoto, K., H. J6rnvall, T. J. MacDonald, M. Carlquist, V. L. W. Go, C. Johansson and V. Mutt. Isolation and primary structure of human PHI (peptide HI). FEBS Lett 174: 258-261, 1984. 24. Wallis, C., J. L. Melnick and C. P. Gerba. Concentration of viruses from water by membrane chromatography. Anntt Rev Microbiol 33: 413-437, 1979. 25. Yanaihara, N., M. Sakagami, H. Sato, K. Yamamoto, T. Hashimoto, C. Yanaihara, Z. lto, K. Yamaguchi and K. Abe. Immunological aspects of secretin, substance P, and V1P. Gastroenterology 72: 803-810, 1977. 26. Yanaihara, N., S. Kobayashi, H. Sato, C. Yanaihara, M. Sakagami, N. Sakura, T. Hashimoto, S. lshikawa, T. lwanaga and T. Fujita. Vasoactive intestinal polypeptide-like immunoreactivity in a human neuroblastoma cell line and the coexistence of other neuropeptide immunoreactivity in the cell line. Endocrinol Jpn 27: Suppl 1, 37-42, 1980. 27. Yanaihara, N., T. Suzuki, H. Sato, M. Hoshino, Y. Okaru and C. Yanaihara. Dibutyryl cAMP stimulation of production and release of VIP-like immunoreactivity in a human neuroblastoma cell line. Biomed Res 2: 728-734, 1981. 28. Yanaihara. N., C. Yanaihara, K. Nokihara, K. Iguchi, S. Fukata, M. Tanaka, Y. Yamamoto and T. Mochizuki. lmmunochemical study on PHI/PHM with use of synthetic peptides. Peptides 5: 247-254, 1984.