Radioimmunoassay of neuropeptide Y

Regulatory Peptides, 8 (1984) 61-70

61

Elsevier RPT00259

Radioimmunoassay of neuropeptide Y J.M. Allen, J.C. Yeats, T.E. Adrian and S.R. Bloom Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 OHS, U.K.

(Received 24 August 1983; revisedmanuscriptreceived2 October 1983; accepted for publication23 November1983)

Summary The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for deterrmnatton of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences. D

•

•

peptide YY; pancreatic polypeptides; reverse phase HPLC; NPY fragments; species specificity

Introduction In 1982, a new putative neurotransmitter was isolated from porcine brain using a novel chemical approach [1,2]. Neuropeptide tyrosine became known by its abbreviation NPY and was found to consist of 36 amino acids, being characterised by an

Address for correspondence: Professor S.R. Bloom, 2nd Floor Francis Fraser Lab., Hammersmith

Hospital, Du Cane Road, London W12 0HS, U.K. 016%0115/84/$03.00 © 1984 Elsevier SciencePublishers B.V.

62 N-terminal tyrosine (Y) and C-terminal tyrosine amide. This new peptide was found to possess remarkable amino acid sequence homology with peptide YY (PYY) [3] and other members of the pancreatic polypeptide (PP) family of peptides. Since its isolation, NPY has been found to possess potent vasoconstrictor activity [4,5] and has been postulated to act as a neuromodulator of noradrenergic transmission [6,7]. The aim of this study was to develop a radioimmunoassay for NPY.

Methods Immunisation Four immunisation schedules were employed to raise antisera to NPY. For each schedule three lop-eared rabbits were immunised by four subcutaneous injections into each groin and axilla. The first 3 schedules used NPY (gift from Dr. Tatemoto, Karolinska Institute) conjugated to bovine serum albumin (BSA, Armour Pharmaceuticals) in a molar ratio of 4 to 1. The third and subsequent boosts used keyhole limpet haemocyanin as carrier. Three standard conjugation reactions were employed, carbodiimide, glutaraldehyde and bis-diazotised benzidine [8]. In addition, a fourth schedule employed unconjugated N'PY iSynthetic, Bachem, U.S.A.) (Table I). The primary injection consisted of 10 nmol conjugated NPY (or 24 nmol unconjugated NPY) in 2 ml complete Freund's adjuvant. After three months, boosts of 5 nmol conjugated (or 12 nmol unconjugated) NPY were given at monthly intervals in incomplete Freund's adjuvant. Rabbits were bled from a marginal ear vein 7 and 10 days after each boost. All antisera were assessed for titre, avidity of binding for 12~I-NPY and the degree of displacemen~of antibody bound 125I-NPY by standard NPY. The specificity of each antiserum was tested by adding up to 100 pmol PYY, avian PP (APP), human PP (HPP), bovine PP'(BPP) and porcine PP (PPP). The ability of three fragments of NPY obtained after trypsin digestion (gift from Dr. Tatemoto) to displace 125I-NPY binding of the four antisera chosen was determined. These fragments consisted of T1 1-19, T220-25 and T3 26-33.

TABLE I NPY immunisationregimesfor 12 rabbits Rabbits YN1,2,3 YN4,5,6 YN7,8,9 YN10,11,12

Couplingagent Carbodiimide Glutaraldehyde Bisdiazotised benzidine Direct immunisation

Primary 10 nmol 10 nmol 10 nmol

Boosts 5 nmol× 4 5 nmol× 4 5 nmolx 4

24 nmol

12 nmol

63

"~'~" ~ . . . . . . . . . . . . pmol per ml

~.~'~

L35%

NPY 0

r 45%

PYY~ I

5.0

0

t porcineextract

i

6.0 human brain extract

0

I

0

I

10

I

ml

20

30

Fig. 1. Radioimmunoassay of 1-ml fractions of (a) porcine NPY and PYY standards, (b) porcine tissue extract (brain), and (c) human brain using non-specific antiserum YN1.

Iodination NPY (2 nmol) was iodinated using chloramine T oxidation with 0.4 nmol Na125I (Amersham 1MS 30) and 70 nmol chloramine T. After 10 s, the reaction was terminated by the addition of 200 nmol sodium metabisulphite. Labelled NPY was purified on an HPLC Bondapak C-18 reverse phase column (3.9 × 300 mm), equilibrated at 35% acetonitrile in water containing 0.2% trifluoroacetic acid. After 30 min, the column was eluted with 405£ acetonitrile in water containing 0.2% trifluoroacetic acid. The flow rate was 1 ml per rain and 1-ml fractions were collected and radioactivity of each fraction assessed. The specific activity of the label was calculated using self displacement. Assay conditions Assays were set up in duplicate polystyrene tubes each in a final volume of 800 #1. Several parameters were investigated to optimise the assay for NPY for each antibody and these included the nature of the buffer, incubation time and preincubation in the absence of labelled NPY. Various buffers of different pH were investigated including sodium acetate buffers (0.1 M) at pH 5.0 and pH 6.0, phosphate buffer (0.06 M) pH 7.2, and sodium barbitone buffer (0.05 M) pH 8.0. All buffers contained 7.7 mM sodium

64

pmol per ml

~'~ .............

t" ot

F/ 45=I°

standards

0

I"

'1

porcine extract

5.0

I

6.0

0

t

!

human brain extract

) 0

i 10

ml

i 20

i 30

Fig. 2. As for Fig. 1 using specific antiserum YN7.

azide and a minimum of 0.3% BSA (43 mM). In addition the effect of increasing the concentration of BsA in the phosphate buffer to 1% and 2% was inves[igated. . After incubation for 5-9 days, the antibody bound and free NPY were separated by the addition of 4 mg charcoal (Norit GSX, Hopkin and Williams) with 400 #g Dextran (7d7 Sigma) in 0.25 ml assay buffer. After centrifuption (800 × g at 4°C for 20 rain) the siipcrnatant was separated from the charcoal pellet and both the free and bound fractions were counted on a gamma counter.

Chromatographf Fractionation of NPY-like immunorcactivity was performed by HPLC using a Bondapak C-18 reverse phase column (3.9 × 300 mm). The column was ¢luted at a flow rate of 2 ml per rain and equilibrated at 35% acetonitrile in water containing 0.2% trifluoroacetic acid. Following the addition of the sample, a linear gradient was used from 35% to 45% acetonitrile in water containing 0.2% trifluoroacefic acid over 10 rain. Porcine NPY and PYY were used as standards in runs before and after the unknowns (Fig. 1).

Tissue extraction Weighed tissues were extracted by boiling in 0.5 M acetic acid (10% weight/volume) for 10 rain. Extracts were stored at - 2 0 ° C until assay. All tisstm peptide concentrations were expressed as means and standard error of the means (S.E.M.).

65 Results

A number of rabbits produced antibodies to NPY with all the various immunisation schedules. Thus two of the three rabbits immunised with carbodiimide and glutaraldehyde conjugated NPY and all three rabbits immunised with the bis-diazotised benzidine conjugates and with unconjugated NPY preparations, produced antisera to NPY. Of these antisera, four were selected for further investigation (Table II). The optimal buffer for all antibodies was found to be phosphate buffer (0.06 M), pH 7.2, containing 1% BSA. Both antisera raised to carbodiimide conjugated NPY showed cross-reaction with the related peptide PYY. Antibody YN3 demonstrated complete cross-reactivity to PYY whereas YN1 only demonstrated 30% cross-reaction. Both the antisera raised to the glutaraldehyde conjugated NPY were absolutely specific for NPY showing no cross-reaction in assay with the related peptides (PYY, APP, HPP, PPP, BPP). All three antisera raised to bisdiazotised benzidine conjugated NPY were specific to NPY up to 10 pmol addition of PYY. None of these antisera showed cross reaction with APP, or PPP up to 100 pmol addition. All three antisera raised by direct immunisation with synthetic NPY showed 5% cross-reaction with APP and 50% cross-reaction with PYY but no cross-reaction with PPP or BPP.

.............

pmolperml .~,~'~'~'~'~'~ 5.0 .~.~'~'~'~ t

45%

35~

standards 0 5.0 I

0

porcineextract

J

6.0 human brain extract

0

0

I

]0

I

ml

20

Fig. 3. As for Fig. 2 using specific antiserum YN6.

30

Conjugation--

CDI

Glutaraldehyde

Bisdiazotised benzidine

Direct immunisation

Antiserum

YN1

YN6

YN7

YN10

1st

4th

4th

4th

Boost

1 : 10000

1 : 400

1 : 2 000

1 : 10000

Final titre

Characteristics of four antibodies used for RIA of NPY

TABLE II

3.1

0.9

1.0

1.2

K value ( × 10 t° 1- m o l - 1)

C-terminal 27-36

N-terminal 1 - 19

-

C-terminal 27-36

Region specificity (fragment binding)

30% cross-reaction with PYY and 10~ with APP. N o significant cross-reaction with HPP or BPP up to 100 pmol per assay tube

No significant cross-reaction with PYY, APP, HPP up to 100 pmol peptide per assay tube

No significant cross-reaction with PYY, APP, HPP or BPP up to 100 pmol peptide per assay tube

30~ with PYY. No significant cross-reaction with APP

Cross-reactivity

67

Only antiserum YN7 showed any significant cross-reactivity with the N-terminal fragments of NPY, binding the T 1 fragment. The other antibodies showed no cross-reactivity with any of the fragments investigated. The two non-specific antibodies YN1 and YN10 were tested against C-terminal fragments of the related peptide PYY and binding was displaced by the C-terminal fragment, 27-36. lodination of NP Y NPY tracer had a specific activity of 70 Bq/fmol. The label, stored in 50% acetonitrile in water (0.2% trifluoroacetic acid) at -20°C, showed no significant decline in immunoreactivity over three months. Assay conditions Phosphate buffer'0.06 M pH 7.2 containing 1% BSA was found to be optimal for all antisera, though differences were not great. Using these four antisera, the concentration of NPY was determined in tissue from six different mammalian species. Only the porcine brain extracts showed reproducible results with all antisera employed (Table III). Using extracts from the other five species, three antisera (YN1, YN7 and YN10) produced remarkably consistent results, whereas the fourth antiserum, YN6, undervalued all these species to different extents.

T A B L E III Concentrations of NPY: p m o l / g wet weight in six species using four antibodies of different specificity Species

YN1

YN10

YN7 -

YN6

Porcine brain (n 4)

44.7 4- 5.7

38.7 + 3.1

39.3 ± 3.2

Human hypbthalamus

92.0 4- 6.7

107.6 4- 8.2

104.2 4- 6.9

20.1 4-1.0

103.0 4-15.2

125.9 4-11.6

117.9 4-17.6

24.2 4- 7.9

50.7 + 4.7

54.1 4- 9.2

54.8 4- 8.4

5.2 + 1.5

Cat spinal cord (n = 3)

8.5 4- 2.1

7.9 4-1.3

8.2 4-1.0

Mouse whole brain (n = 5)

77.7 4-11.1

82.9 4- 4.9

70.8 4- 5.9

=

(. = 5) Rat hypothalamus

(n =3) G u i n e a pig spinal cord

(,=5)

Values are m e a n + S.E.M.

U.D. 12.1 4- 2.9

68

Chromatography Using HPLC the two structurally related peptides PYY and NPY were separated. Porcine PYY standard duted from the gradient at 36% acetonitrile in water (0.2% trifluoroaeetic acid) and NPY standard consistently was eluted at 45% acetonitrile in water (0.2% trifluoroacetic acid). Recoveries of the applied standards were consistently above 85% using all four antisera to assay the fractions. In keeping with their specificity, YN6 and YN7 could not detect the PYY standard. Porcine tissue extract eluted in the position of NPY standard. Cat, guinea pig, mouse and rat tissue extracts similarly coeluted in the position of porcine NPY whereas the NPY-like immunoreactivity in human tissue extracts consistently duted from the column earlier than porcine NPY standard, and this was observed with all human tissue extracts applied to the column (brain regions, spinal cord and adrenal). In order to ensure that the difference in concentrations observed in the tissue extracts was not due to different molecular forms of NPY, the column fractions were assayed using three antibodies YN1, YN6 and YN7. No different molecular species were seen by these three antibodies, but the absolute level within each fraction differed in keeping with the difference observed in the original tissue extract.

Discussion

Neuropeptide Y (NPY) is the most recently discovered member of the pancreatic polypeptide (PP) family of peptides. Although isolated from porcine brain [1,2], this peptide has been identified within neurones outside the central nervous system [6,9], and has been shown to possess potent vasoconstrictor activity [4,5]. The development of a radioimmunoassay to NPY has allowed determination of its presence and distribution throughout mammalian species and has also been of use in determining its possible physiological role [10]. The different methods of raising antisera to NPY were all successful, although most of the antisera raised had relatively low binding affinity constants. NPY has a remarkable amino acid sequence homology with PYY. Both peptides consist of 36 amino acids and possess N-terminal tyrosine and C-terminal tyrosine amide residues. Only 11 amino acids differ between the two peptides and substitutions are, in general, conservative. The N-terminal region of the two peptides bears the major differences and thus it would be expected that a specific antibody would be directed towards this end. This was indeed found to be the case with the antiserum YN7, which is specific for NPY. The non-specific antibodies, such as YN1 and YN10 conversely could be predicted to be directed towards the C-terminal region as these are almost identical between the two peptides. YN1 and YN10 were found to bind C-terminal fragments of the related peptide, PYY. YN6, the fourth antiserum, is similar to YN7 in that it is absolutely specific, but no binding of the N-terminal or mid-molecule fragments was observed and it is possible that this antiserum is dependent on the correct tertiary structure of the whole molecule for binding. The NPY concentrations in tissues of non-porcine species were always underestimated by this antiserum (YN6) in comparison to the three alternative

69 antisera (YN1, Y N 1 0 and Y N 7 ) and only the porcine tissue extract concentrations for Y N 6 m a t c h e d those of the other three antisera. Members of the pancreatic polypeptide (PP) family vary considerably in amino acid sequence [11,12] and it would therefore not be surprising if this new related m e m b e r was similarly f o u n d to possess significant species variability. All N P Y concentrations extracted from nonporcine species were under read by the Y N 6 antibody, but H P L C of the tissue extracts f r o m four species, guinea pig, mouse, rat and cat showed that the NPY-like immunoreactivity eluted from the column in the position of the porcine standard. The finding that the NPY-like immunoreactivity f r o m extracts of h u m a n tissue consistently eluted from the H P L C column earlier than either porcine N P Y standard or porcine extracts and the difference in antisera specificity for both extracts and fractions provides strong evidence for a h u m a n form of N P Y distinct from porcine NPY.

Acknowledgements Natural porcine NPY, P Y Y and trypsin fragments of N P Y were generously provided b y Dr. K. Tatemoto; Dr. J. Kimmell kindly provided A P P and H P P and Professor Yanaihara d o n a t e d the C-terminal fragments of PYY. J.M.A. is in receipt of a Wellcome Trust Training Fellowship.

References 1 Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature, 296 (1982) 659-660. 2 Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5485-5489. 3 Tatemoto, K., Isolation and ¢haracterisation of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 2514-2518. 4 Lundberg, J.M. and Tatemoto, K., Pancreatic polypeptide family (APP, BPP, NPY & PYY) in relation to sympathetic vasoconstriction resistant to alpha adrenoceptor blockade, Acta Physiol. Stand., 116 (1982) 393-402. 5 Allen, J.M., Bircham, P.M.M., Edwards, A.V., Tatemoto, K. and Bloom, S.R., Neuropeptide Y (NPY) reduces myocardial perfusion and inhibits the force of contraction of the isolated perfused rabbit heart, Regul. Peptides, 6 (1983) 247-254. 6 Lundberg, J.M., Terenius, L., H6kfelt, T., Marthing, C.R., Tatemoto, K., Mutt, V., Polak, J., Bloom, S.R. and Goldstein, M., Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurones and effects of NPY on sympathetic function, Acta Physiol. Stand., 116 (1982) 477-480. 7 Allen, J.M., Adrian, T.E., Tatemoto, K., Polak, J.M., Hughes, J. and Bloom, S.R., Two novel related peptides, neuropeptide Y (NPY) and peptide YY (PYY) inhibit the contraction of the electrically stimulated mouse vas deferens, Neuropeptides, 3 (1982) 71-77. 80'Shaughnessy, D.J., Antibodies. In Bloom, S.R. and Long, R.G. (Eds.), Radioimmunoassay of Gut Regulatory Peptides, W.B. Saunders Company Ltd., London, 1982, pp. 11-20. 9 Gu, J., Polak, J.M., Adrian, T.E., Allen, J.M., Tatemoto, K. and Bloom, S.R., Neuropeptide tyrosine (NPY) - A major new cardiac neuropeptide, Lancet, ii (1983) 1008-1010. 10 Allen, J.M., McGregor, G.P., Adrian, T.E., Bloom, S.R., Zhang, S.Q., Ennis, K.W. and Unger, W.G., Reduction of neuropeptide Y (NPY) in the rabbit iris ciliary body following sympathectomy, Exp. Eye Res., 37 (1983) 213-215.

70 11 Kimmel, J.R., Hayden, L.J. and Pollack, H.G.J., Isolation and charactefisation of new pancreatic polypeptide hormone, J. Biol. Chem., 250 (1975) 9369-9376. 12 Lin, T.M. and Chance, R.E., In Chey, W.Y. and Brooks, F.P. (Eds.), Endocrinology of the gut, Slack Thorofare, N.J., 1974, pp. 143-145.