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Radioimmunoassay to determine postprandial changes in plasma Neuropeptide Y levels in awake dogs
0143-4179/89/0014-0.$10.00
Neuropeptides (1989) 14,209-212 0 LongmanGroupUK Ltd 1989
Radioimmunoassay to Determine Postprandial Changes in Plasma Neuropeptide Y Levels in Awake Dogs A. BALASUBRAMANIAM, D. W. MCFADDEN, M. RUDNICKI, M. S. NUSSBAUM, R. DAYAL, L. S. SRIVASTAVA, and J. E. FISCHER Departments of Surgery and Internal Medicine, 45267, USA (reprint request to ABI
University of Cincinnati Medical Center, Cincinnati, Ohio
Abstract-A specific, precise and sensitive double-antibody radioimmunoassay for Neuropeptide Y (NPY) has been developed. There was no appreciable cross-reactivity with the structurally related peptides, peptide YY (PW) and pancreatic polypeptide (PP). The minimum detectable plasma NPY level was 3nM. Application of radioimmunoassay to canine models revealed that portal and systemic NPY levels increased significantly following a standard meal.
Introduction Neuropeptide Y (NPY) and peptide YY (PYY) are two homologous peptides isolated from the brain (1) and intestine (2), respectively. Immunohistochemical studies have shown that NPY is present throughout the body of mammals in the neurons of the central and peripheral nervous systems (3) while PYY is confined to the endocrine cells of the gut and pancreas (4). Subsequently PYY has been shown to be present in rat brain (5). Sequence homology and overlapping of the physiological functions of these hormones with one another and with pancreatic polypeptide (PP) (6) has led to the classification of these peptides into a new family of homologous hormones.
Our efforts towards studying the tissue distribution and plasma level of these hormones has previously resulted in the development of a sensitive radioimmunoassay for PYY (7). Since the isolation of NPY, most studies have focused on its tissue distribution with only a few reports appearing that describe plasma NPY levels in mammals under various physiological conditions (8-13). In this paper, we describe the development of a sensitive radioimmunoassay for NPY and its application for the first time, to measure postprandial changes in NPY plasma levels in awake dogs.
Materials and Methods Antibody Preparation
Female albino New Zealand rabbits weighing about 2Skg were immunized with porcine NPY
Date received 16 May 1989 Date accepted 16 May 1989
209
210
NEUROPEPT’IDES
600
Assay
r
-
2
6
10
14
16
22
FRACTION
26
30
34
36
42
‘6
a0
50
NUMBER
Fig. 1 Purification of “‘1-NPY by analytical HPLC. Conditions: Vydac C4 column (250 x 4.6mm, 5t~rn particle size), solution A is 0.1% TFA in Ha0 and solution B is 60% CHsCN in A; flow rate is 1 mlknin. The iodination mixture was loaded on to the HPLC column and subjected to a gradient as shown in the Figure. lml fractions were collected and 5~1 of each fraction was counted for radioactivity.
(14) or NPY conjugated to succinylated albumin (15) emulsified with complete Freund’s adjuvant for primary immunization. Injections were given directly into popliteal lymph nodes and also intradermally in multiple sites on the back. Booster injections on the back were given twice approximately three weeks apart with either antigen emulsified with incomplete Freund’s adjuvant. Each injection contained 1OOpg of the antigen. The animals were bled between lo-14 days after each booster injection and the serum frozen at -20°C. Since the antibody raised against NPY conjugated to succinylated albumin cross-reacted with PYY and PP, all our investigations were carried out with antisera raised against free NPY. Iodination Synthetic NPY was iodinated by the chloramine-T method according to published procedures (16). The reaction mixture was loaded onto a reversed phase C4 column and purified as described in Figure 1 legend. The major peak fractions were tested for binding to antibody and the fraction exhibiting highest binding with low non-specific binding was used in the assay.
O.OlM sodium phosphate, pH 7.6 buffer containing 0.15N sodium chloride, O.OlM EDTA, merthiolate (0.1 g/l) and 1% BSA was used as the assay buffer. The following procedure was used: A mixture consisting of 100~1 sample or standard, lOO$ of assay buffer or NPY free plasma and 100 ~1 NPY antiserum (1:2000 initial dilution) was incubated overnight (=20h) at 4°C. This was followed by the addition of 100 l.~l1251-NPY tracer (10000 CPM per tube) and overnight (= 20 h) incubation at 4°C. lOOl.rl of antirabbit gamma globulin and 100 ~1 10% polyethylene glycol were then added, and the mixture incubated at ambient temperature for 2 h. 500 l~,lof 1% BSA assay buffer were added, and the bound and free ‘251-NPY were separated by centrifugation at 3000 rpm for 20 min. Supernatant was aspirated and the residue in the tube was counted for 5 min. The result was calculated by using logit log transformation of the data on a Wang computer. Plama sample preparation lml of plasma was extracted with 1.6 vol of 95% ethanol and the supernatant fluid obtained after centrifugation was air dried. The residue was dissolved in lml of assay buffer. After removing undissolved material by centrifugation, the supernant fluid was assayed at various levels of dilution. For reversed phase chromatography experiments 3ml of plasma was extracted similarly. Chromotography experiments were carried out according to published procedures (13). Postprandial release of NPY Conditioned dogs (n = 6) weighing about 25 kg with both portal and peripheral venous catheters were used in the investigation (17). After overnight fasting, with ad libitum access to fresh water, both portal and systemic blood samples were collected to estimate basal NPY levels. A standard meal (425g) was then given and blood samples were collected at the end of each 20 min period for 2 h. Five ml blood samples were collected in chilled glass tubes containing 2500 KIU of aprotinin and 100~ of Heparin and the plasma samples obtained by centrifugation were stored at -20°C until assayed.
RADIOIMMUNOASSAY
0
5
10
TO DETERMINE POSTPRANDIAL CHANGES IN PLASMA NEUROPEPTIDE Y LEVELS
II FRACTION
20
25
30
35
40
NUMBER
Fig. 2 Rechromatography of the main NPY-like immunoreactivity peak present in the systemic plasma of dog. Chromatography conditions were same as in Figure 1. Plasma was extracted as described in materials and methods and the main NPY-like immunoreactivity peak in the plasma was isolated using identical conditions. Arrow indicates the elution position of synthetic NPY.
Results After extensive evaluation, conditions found optimal for antibody-antigen interaction were used in this investigation as described in materials and methods. The tracer used in this study was obtained by reversed phase chromatography of the iodination reaction mixture. HPLC resulted in one major peak and one minor peak (Fig. 1). The
minor peak exhibited very little binding. Investigations with fractions 23,24,25 and 26 showed that fraction 25 had the highest binding (45%) and lowest nonspecific binding (<5%), and therefore this 12’I-NPY fraction was used throughout the study. Under the conditions employed, the minimum detection limit of NPY was 3nM. Interassay and intrassay coefficient of variations were 9.58 and 9.33%, respeclively. Recoveries of NPY standard was usually > 95%. Antisera had no cross-reactivity (18) with secretin, peptide Histidine-Isoleucine, vasoactive intestinal peptide, and NPY fragments l-23 and 17-36. Furthermore, antisera showed no appreciable cross-reactivity with the structurally-related peptides, PYY and PP (Table). Reversed phase chromatography of canine plasma extracts showed that the major NPY-like immunoreactivity eluted at the same time as the standard (Fig. 2). There was no significant difference in the basal levels of NPY in canine portal and systemic plasma samples (Fig. 3). Ingestion of the standard meal caused a significant increase in NPY levels in both portal and systemic plasma in the first 20 min period. NPY levels fell afterwards and reached the basal state in about 100 min. Discussion The results presented in this paper show that our radioimmunoassay is sensitive, precise and highly specific for NPY. Furthermore, the failure of our 550
Table Cross-Reactivity of Various Peptides with NPY Antisera Peptide
NPY NPY (l-23) NPY (17-36) PYY PP Secretin VIP PHI
% Cross Reactivity”
100 0 0 1.4 1.1 0 0 0
a Cross-reactivity is based on the relative concentrations of Peptide and of NPY needed to inhibit 50% of the binding of ‘*‘I-NPY to antiserum. Peptides which did not inhibit 12sI-NPY to antiserum up to 1OOpmoUtube is given a cross reactivity value of zero.
211
1
500
4 I
450
I h
400
350 lime (nun) Fig. 3 Portal (Cl) and systemic (W) plasma NPY level in awake dogs before and after ingestion of standard meal. (mean + SEM; *p < 0.05 vs. basal by repeated measures of ANOVA).
212 antisera to recognize the partial sequences of NPY, l-23 and 17-36, suggest that it may be dependent on the tertiary structure of the entire NPY molecule for binding. Reversed phase chromatography experiments show that the major immunoreactive peak present in canine plasma extracts corresponds to the same molecular form as intact NPY. Application of radioimmunoassay to canine models revealed that a standard meal caused an elevation of NPY level in both portal and systemic plasma. This simultaneous increase in NPY levels in portal and systemic plasma suggests that the source of this NPY may be from the gastrointestinal tract. Furthermore, we have recently found that there is a decrease in ileal luminal NPY following a protein meal in dogs (17). It may therefore be suggested that ingestion of food may trigger the release of NPY into portal and hence, systemic plasma. In conclusion, a sensitive and specific radioimmunoassay for NPY has been developed and applied for the first time to determine the basal as well as postprandial changes of plasma NPY level in canine. Canine models have been extensively used in understanding the postprandial release of other hormones such as PYY and PP, and therefore the radioimmunoassayof NPY described in this paper for canine models, is now being expanded in our laboratory to study the effects of other nutrients on the postprandial release of NPY into the plasma and intestinal lumen, This investigation may lead to a better understanding of the mechanisms of postprandial release of NPY.
NEUROPEPHDES
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Acknowledgement This work was supported in part by NIHgrant GM 38601 (AB). We thank T. Linton and L. Carovillano for secretarial support.
References 1. Tatemoto, K. (1982). Neuropeptide Y; complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A. 79: 54855489. 2. Tatemoto, K. (1982). Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion. Proc. Natl. Acad. Sci. U.S.A. 79: 2514-2518. 3. Lundberg, J. M., Terenius, L., Hokfelt, T., Martling, C. R., Tatemoto, K., Mutt, V. Polak, J., Bloom, S. and Goldstein, M. (1982). Neuropeptide Y (NPY)-like
14.
15.
16.
17.
18.
Immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function, Acta Physiol. Stand. 116: 477-480. Lundberg, J. M., Tatemoto, K., Terenius, L., Hellstrom, P. M., Mutt, V. Hokfelt, T. and Hamberger, B. (1982). Localization of peptide YY (PYY) in gastrointestinal endocrine cells and effects on intestinal blood flow and motility. Proc. Natl. Acad. Sci. U.S.A. 79: 4471-4475. Ekman, R., Wahlestedt, C., Bottchen, G., Sundler, F., Halanson, R. and Pamela, P. (1986). Peptide YY-like immunoreactivity in the central nervous system of rat. Regul. Pept. 16: 157- 159. Kimmel,J.R.,Hayden,L.J.andPollock,H.G.J.(1975). Isolation and characterization of new pancreatic polypeptide hormone, J. Biol. Chem. 250: 9369-9376. Chen, M-H., Balasubramaniam, A., Murphy, R. F., Tabata, K., Fischer, J. E., Chen, I-W. and Joffe, S. N. (1984). Sensitive radioimmunoassay for measurement of circulating peptide YY, Gastroenterology 87: 1332-1338. Allen, Y. S., Adrian, T. E., Allen, J. M., Tatemoto, K., Crow, T. J., Bloom, S. R. and Polak, J. M. (1983). Neuropeptide Y distribution in the rat brain. Science 221: 877-879. Allen, J. M., Yeats, J. C., Adrian, T. E. and Bloom, S. R. (1984). Radioimmunoassay of Neuropeptide Y. Reg. Pept. 8: 61-70. Everitt, B. J., Hokfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M. (1984). Differential coexistence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat. Neuroscience 11: 443-462. Uddman, R., Sundler, F. and Emson, P. (1984). Occurrence and distribution of neuropeptide-Y-immunoreactive nerves in the respiratory tract and middle ear. Cell Tissue and Res. 237: 321-327. Lundberg, J. M., Martinsson, A., Hemsen, A., Theodorsson-Norheim, E., Svedenhag, J., Ekblom, B. and Hjemdahl, P. (1985). Biochem. Biophys. Res. Commun. 133: 30-36. McDonald, J. K., Koenig, J. I., Gibbs, D. M., Collins, P. and Noe, B. D. (1987). High concentrations of neuropeptide Y in pituitary portal blood of rats. Neuroendocrinology 46: 538-541. Balasubramaniam, A., Grupp, I. L., Srivastava, L., Tatemoto, K., Murphy, R. F., Joffe, S. N. and Fischer, J. E. (1987). Synthesis of Neuropeptide Y. Int. J. Peptide Protein Res. 29: 78-82. Ebid, A. M., Murray, P., Soeters, P. B. and Fischer, J. E. (1977). Release of VIP by calcium stimulation. Am. J. Surg. 133: 140-144. Hunter, W. M. and Greenwood, F. C. (1962). Preparation of iodine-131 human growth hormone of high specific activity. Nature 194: 495-496. Rudnicki. M.. McFadden, D. W., Balasubramaniam, A., Nussbaum, M. S.. Fischer, J. E. (1989). The Postprandial Circulatory and Ileal Intraluminal Release of Neuropeptide Y in Conscious Dogs, submitted. Abraham, G. E. (1969). Solid-phase radioimmunoassay of 8-estradiol. J. Clin. Endocrin. 39: 866-870.
Neuropeptides (1989) 14,209-212 0 LongmanGroupUK Ltd 1989
Radioimmunoassay to Determine Postprandial Changes in Plasma Neuropeptide Y Levels in Awake Dogs A. BALASUBRAMANIAM, D. W. MCFADDEN, M. RUDNICKI, M. S. NUSSBAUM, R. DAYAL, L. S. SRIVASTAVA, and J. E. FISCHER Departments of Surgery and Internal Medicine, 45267, USA (reprint request to ABI
University of Cincinnati Medical Center, Cincinnati, Ohio
Abstract-A specific, precise and sensitive double-antibody radioimmunoassay for Neuropeptide Y (NPY) has been developed. There was no appreciable cross-reactivity with the structurally related peptides, peptide YY (PW) and pancreatic polypeptide (PP). The minimum detectable plasma NPY level was 3nM. Application of radioimmunoassay to canine models revealed that portal and systemic NPY levels increased significantly following a standard meal.
Introduction Neuropeptide Y (NPY) and peptide YY (PYY) are two homologous peptides isolated from the brain (1) and intestine (2), respectively. Immunohistochemical studies have shown that NPY is present throughout the body of mammals in the neurons of the central and peripheral nervous systems (3) while PYY is confined to the endocrine cells of the gut and pancreas (4). Subsequently PYY has been shown to be present in rat brain (5). Sequence homology and overlapping of the physiological functions of these hormones with one another and with pancreatic polypeptide (PP) (6) has led to the classification of these peptides into a new family of homologous hormones.
Our efforts towards studying the tissue distribution and plasma level of these hormones has previously resulted in the development of a sensitive radioimmunoassay for PYY (7). Since the isolation of NPY, most studies have focused on its tissue distribution with only a few reports appearing that describe plasma NPY levels in mammals under various physiological conditions (8-13). In this paper, we describe the development of a sensitive radioimmunoassay for NPY and its application for the first time, to measure postprandial changes in NPY plasma levels in awake dogs.
Materials and Methods Antibody Preparation
Female albino New Zealand rabbits weighing about 2Skg were immunized with porcine NPY
Date received 16 May 1989 Date accepted 16 May 1989
209
210
NEUROPEPT’IDES
600
Assay
r
-
2
6
10
14
16
22
FRACTION
26
30
34
36
42
‘6
a0
50
NUMBER
Fig. 1 Purification of “‘1-NPY by analytical HPLC. Conditions: Vydac C4 column (250 x 4.6mm, 5t~rn particle size), solution A is 0.1% TFA in Ha0 and solution B is 60% CHsCN in A; flow rate is 1 mlknin. The iodination mixture was loaded on to the HPLC column and subjected to a gradient as shown in the Figure. lml fractions were collected and 5~1 of each fraction was counted for radioactivity.
(14) or NPY conjugated to succinylated albumin (15) emulsified with complete Freund’s adjuvant for primary immunization. Injections were given directly into popliteal lymph nodes and also intradermally in multiple sites on the back. Booster injections on the back were given twice approximately three weeks apart with either antigen emulsified with incomplete Freund’s adjuvant. Each injection contained 1OOpg of the antigen. The animals were bled between lo-14 days after each booster injection and the serum frozen at -20°C. Since the antibody raised against NPY conjugated to succinylated albumin cross-reacted with PYY and PP, all our investigations were carried out with antisera raised against free NPY. Iodination Synthetic NPY was iodinated by the chloramine-T method according to published procedures (16). The reaction mixture was loaded onto a reversed phase C4 column and purified as described in Figure 1 legend. The major peak fractions were tested for binding to antibody and the fraction exhibiting highest binding with low non-specific binding was used in the assay.
O.OlM sodium phosphate, pH 7.6 buffer containing 0.15N sodium chloride, O.OlM EDTA, merthiolate (0.1 g/l) and 1% BSA was used as the assay buffer. The following procedure was used: A mixture consisting of 100~1 sample or standard, lOO$ of assay buffer or NPY free plasma and 100 ~1 NPY antiserum (1:2000 initial dilution) was incubated overnight (=20h) at 4°C. This was followed by the addition of 100 l.~l1251-NPY tracer (10000 CPM per tube) and overnight (= 20 h) incubation at 4°C. lOOl.rl of antirabbit gamma globulin and 100 ~1 10% polyethylene glycol were then added, and the mixture incubated at ambient temperature for 2 h. 500 l~,lof 1% BSA assay buffer were added, and the bound and free ‘251-NPY were separated by centrifugation at 3000 rpm for 20 min. Supernatant was aspirated and the residue in the tube was counted for 5 min. The result was calculated by using logit log transformation of the data on a Wang computer. Plama sample preparation lml of plasma was extracted with 1.6 vol of 95% ethanol and the supernatant fluid obtained after centrifugation was air dried. The residue was dissolved in lml of assay buffer. After removing undissolved material by centrifugation, the supernant fluid was assayed at various levels of dilution. For reversed phase chromatography experiments 3ml of plasma was extracted similarly. Chromotography experiments were carried out according to published procedures (13). Postprandial release of NPY Conditioned dogs (n = 6) weighing about 25 kg with both portal and peripheral venous catheters were used in the investigation (17). After overnight fasting, with ad libitum access to fresh water, both portal and systemic blood samples were collected to estimate basal NPY levels. A standard meal (425g) was then given and blood samples were collected at the end of each 20 min period for 2 h. Five ml blood samples were collected in chilled glass tubes containing 2500 KIU of aprotinin and 100~ of Heparin and the plasma samples obtained by centrifugation were stored at -20°C until assayed.
RADIOIMMUNOASSAY
0
5
10
TO DETERMINE POSTPRANDIAL CHANGES IN PLASMA NEUROPEPTIDE Y LEVELS
II FRACTION
20
25
30
35
40
NUMBER
Fig. 2 Rechromatography of the main NPY-like immunoreactivity peak present in the systemic plasma of dog. Chromatography conditions were same as in Figure 1. Plasma was extracted as described in materials and methods and the main NPY-like immunoreactivity peak in the plasma was isolated using identical conditions. Arrow indicates the elution position of synthetic NPY.
Results After extensive evaluation, conditions found optimal for antibody-antigen interaction were used in this investigation as described in materials and methods. The tracer used in this study was obtained by reversed phase chromatography of the iodination reaction mixture. HPLC resulted in one major peak and one minor peak (Fig. 1). The
minor peak exhibited very little binding. Investigations with fractions 23,24,25 and 26 showed that fraction 25 had the highest binding (45%) and lowest nonspecific binding (<5%), and therefore this 12’I-NPY fraction was used throughout the study. Under the conditions employed, the minimum detection limit of NPY was 3nM. Interassay and intrassay coefficient of variations were 9.58 and 9.33%, respeclively. Recoveries of NPY standard was usually > 95%. Antisera had no cross-reactivity (18) with secretin, peptide Histidine-Isoleucine, vasoactive intestinal peptide, and NPY fragments l-23 and 17-36. Furthermore, antisera showed no appreciable cross-reactivity with the structurally-related peptides, PYY and PP (Table). Reversed phase chromatography of canine plasma extracts showed that the major NPY-like immunoreactivity eluted at the same time as the standard (Fig. 2). There was no significant difference in the basal levels of NPY in canine portal and systemic plasma samples (Fig. 3). Ingestion of the standard meal caused a significant increase in NPY levels in both portal and systemic plasma in the first 20 min period. NPY levels fell afterwards and reached the basal state in about 100 min. Discussion The results presented in this paper show that our radioimmunoassay is sensitive, precise and highly specific for NPY. Furthermore, the failure of our 550
Table Cross-Reactivity of Various Peptides with NPY Antisera Peptide
NPY NPY (l-23) NPY (17-36) PYY PP Secretin VIP PHI
% Cross Reactivity”
100 0 0 1.4 1.1 0 0 0
a Cross-reactivity is based on the relative concentrations of Peptide and of NPY needed to inhibit 50% of the binding of ‘*‘I-NPY to antiserum. Peptides which did not inhibit 12sI-NPY to antiserum up to 1OOpmoUtube is given a cross reactivity value of zero.
211
1
500
4 I
450
I h
400
350 lime (nun) Fig. 3 Portal (Cl) and systemic (W) plasma NPY level in awake dogs before and after ingestion of standard meal. (mean + SEM; *p < 0.05 vs. basal by repeated measures of ANOVA).
212 antisera to recognize the partial sequences of NPY, l-23 and 17-36, suggest that it may be dependent on the tertiary structure of the entire NPY molecule for binding. Reversed phase chromatography experiments show that the major immunoreactive peak present in canine plasma extracts corresponds to the same molecular form as intact NPY. Application of radioimmunoassay to canine models revealed that a standard meal caused an elevation of NPY level in both portal and systemic plasma. This simultaneous increase in NPY levels in portal and systemic plasma suggests that the source of this NPY may be from the gastrointestinal tract. Furthermore, we have recently found that there is a decrease in ileal luminal NPY following a protein meal in dogs (17). It may therefore be suggested that ingestion of food may trigger the release of NPY into portal and hence, systemic plasma. In conclusion, a sensitive and specific radioimmunoassay for NPY has been developed and applied for the first time to determine the basal as well as postprandial changes of plasma NPY level in canine. Canine models have been extensively used in understanding the postprandial release of other hormones such as PYY and PP, and therefore the radioimmunoassayof NPY described in this paper for canine models, is now being expanded in our laboratory to study the effects of other nutrients on the postprandial release of NPY into the plasma and intestinal lumen, This investigation may lead to a better understanding of the mechanisms of postprandial release of NPY.
NEUROPEPHDES
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Acknowledgement This work was supported in part by NIHgrant GM 38601 (AB). We thank T. Linton and L. Carovillano for secretarial support.
References 1. Tatemoto, K. (1982). Neuropeptide Y; complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A. 79: 54855489. 2. Tatemoto, K. (1982). Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion. Proc. Natl. Acad. Sci. U.S.A. 79: 2514-2518. 3. Lundberg, J. M., Terenius, L., Hokfelt, T., Martling, C. R., Tatemoto, K., Mutt, V. Polak, J., Bloom, S. and Goldstein, M. (1982). Neuropeptide Y (NPY)-like
14.
15.
16.
17.
18.
Immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function, Acta Physiol. Stand. 116: 477-480. Lundberg, J. M., Tatemoto, K., Terenius, L., Hellstrom, P. M., Mutt, V. Hokfelt, T. and Hamberger, B. (1982). Localization of peptide YY (PYY) in gastrointestinal endocrine cells and effects on intestinal blood flow and motility. Proc. Natl. Acad. Sci. U.S.A. 79: 4471-4475. Ekman, R., Wahlestedt, C., Bottchen, G., Sundler, F., Halanson, R. and Pamela, P. (1986). Peptide YY-like immunoreactivity in the central nervous system of rat. Regul. Pept. 16: 157- 159. Kimmel,J.R.,Hayden,L.J.andPollock,H.G.J.(1975). Isolation and characterization of new pancreatic polypeptide hormone, J. Biol. Chem. 250: 9369-9376. Chen, M-H., Balasubramaniam, A., Murphy, R. F., Tabata, K., Fischer, J. E., Chen, I-W. and Joffe, S. N. (1984). Sensitive radioimmunoassay for measurement of circulating peptide YY, Gastroenterology 87: 1332-1338. Allen, Y. S., Adrian, T. E., Allen, J. M., Tatemoto, K., Crow, T. J., Bloom, S. R. and Polak, J. M. (1983). Neuropeptide Y distribution in the rat brain. Science 221: 877-879. Allen, J. M., Yeats, J. C., Adrian, T. E. and Bloom, S. R. (1984). Radioimmunoassay of Neuropeptide Y. Reg. Pept. 8: 61-70. Everitt, B. J., Hokfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M. (1984). Differential coexistence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat. Neuroscience 11: 443-462. Uddman, R., Sundler, F. and Emson, P. (1984). Occurrence and distribution of neuropeptide-Y-immunoreactive nerves in the respiratory tract and middle ear. Cell Tissue and Res. 237: 321-327. Lundberg, J. M., Martinsson, A., Hemsen, A., Theodorsson-Norheim, E., Svedenhag, J., Ekblom, B. and Hjemdahl, P. (1985). Biochem. Biophys. Res. Commun. 133: 30-36. McDonald, J. K., Koenig, J. I., Gibbs, D. M., Collins, P. and Noe, B. D. (1987). High concentrations of neuropeptide Y in pituitary portal blood of rats. Neuroendocrinology 46: 538-541. Balasubramaniam, A., Grupp, I. L., Srivastava, L., Tatemoto, K., Murphy, R. F., Joffe, S. N. and Fischer, J. E. (1987). Synthesis of Neuropeptide Y. Int. J. Peptide Protein Res. 29: 78-82. Ebid, A. M., Murray, P., Soeters, P. B. and Fischer, J. E. (1977). Release of VIP by calcium stimulation. Am. J. Surg. 133: 140-144. Hunter, W. M. and Greenwood, F. C. (1962). Preparation of iodine-131 human growth hormone of high specific activity. Nature 194: 495-496. Rudnicki. M.. McFadden, D. W., Balasubramaniam, A., Nussbaum, M. S.. Fischer, J. E. (1989). The Postprandial Circulatory and Ileal Intraluminal Release of Neuropeptide Y in Conscious Dogs, submitted. Abraham, G. E. (1969). Solid-phase radioimmunoassay of 8-estradiol. J. Clin. Endocrin. 39: 866-870.