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Distribution and chromatographic characterization of neuromedin B-like immunoreactivity in the human spinal cord
Brain Research, 342 (1985) 183-186 Elsevier
183
BRE 21011
Distribution and chromatographic characterization of neuromedin B-like immunoreactivity in the human spinal cord M. NAMBA, M. A. GHATEI, P. ANAND and S. R. BLOOM Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS ( U. K.
(Accepted April 16th, 1985) Key words: neuromedin B-like immunoreactivity - - spinal cord - - human
The quantitative regional distribution of neuromedin B-like immunoreactivity in normal postmortem human spinal cord was studied by a specific radioimmunoassay. Neuromedin B-like immunoreactivity was found in highest concentration in the dorsal part of the sacral cord. Chromatographic analyses by gel permeation and reverse-phase high-pressure liquid chromatography revealed two major peaks of neuromedin B-like immunoreactivity and the prevalent molecular form, approx. 90% of the total immunoreactivity, was chromatographically identical to synthetic porcine neuromedin B.
N e u r o m e d i n B is a novel d e c a p e p t i d e isolated from porcine spinal cord which shows a striking sequence h o m o l o g y with m e m b e r s of bombesin-like peptide family, particularly the peptides of litorin/ranatensin subfamily, at the C-terminal heptapeptide6. N e u r o m e d i n B shows p o t e n t contractile activity on uterine muscle and stimulatory effects on the release of gastrointestinal and pancreatic regulatory peptides with a profile very similar to that of bombesin6,7. Since the p e p t i d e has potent biological actions, we studied the distribution of n e u r o m e d i n B-like immunoreactivity in human spinal cord. H u m a n spinal cord specimens were collected postmortem from 8 adults (6 males, 2 females; mean 60 year, range 4 8 - 8 3 year) with no evidence of nervous system pathology. All cadavers were stored at 4 °C within 2 h of death, and the spinal cords r e m o v e d within 24 h, a period generally established for stability of n e u r o p e p t i d e s in p o s t m o r t e m specimens 2. For radioimmunoassay, transverse slices (0.5 cm thick) from cervical, thoracic, lumbar and sacral regions were divided into ventral and dorsal regions by section through the central canal and stored in liquid nitrogen. N e u r o m e d i n B was extracted by boiling in 0.5 M acetic acid (10 ml per g wet wt. tissue) for 10
min. Duplicate 20~ul aliquots of each extract were assayed by a specific radioimmunoassay. In order to investigate the efficiency of the extraction, synthetic n e u r o m e d i n B standard was incubated with fresh rat liver h o m o g e n a t e s , for 1 h (n = 4). The mixtures were then boiled in 0.5 M acetic acid for 10 rain. The recovery of exogenous n e u r o m e d i n B-like immunoreactivity after boiling was 102 _+ 6%. The r a d i o i m m u n o a s s a y is described in full elsewhereS. The antiserum (NB 8) was raised in a rabbit to synthetic porcine n e u r o m e d i n B (Cambridge Research Biochemical, Cambridgeshire, U . K . , Lot PMG/22/GIy/3) conjugated to bovine serum albumin (BSA) with glutaraldehyde and used at a final dilution of 1:7000. This antibody cross-reacted 70% with litorin and 60% with ranatensin, but showed no significant cross-reaction with other known neuropeptides including bombesin and substance P. [Try°] neuromedin B (Lot PMG/22/Tyr/5, C a m b r i d g e Research Biochemical) was iodinated by the conventional chloramine-T oxidation. The iodinated product was then purified by high-pressure liquid chromatography ( H P L C ) using a C-18 reverse phase column (10 ~ m , 3.9 x 300 mm) and eluted in 30% acetonitrile in an aqueous solution of 0.1% trifluoroacetic
Correspondence: S. R. Bloom, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London WI2 0HS, U,K.
00(16-8993/85/$(13.3(I © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
184 TABLE
fmol/fraction
1
Regional concentrations of neuromedin B-h'ke immum~reactivttv in human spinal cord
CC
N J';
400
Results are e x p r e s s e d as m e a n + S . E . M . in p m o l / g wet wt.
Cervical Thoracic Lumbar Sacral
Ventral
Dorsal
1.9 2.0 3.9 3.3
2.0 3.6 4.7 12.4
+_ 0.7 + 0.8 + 0.9 _+_0.8
+ (t.4 _+ 1.2 _+ (1.8 ± 1.7
acid. The assay was carried out in 0.06 M phosphate buffer, p H 7.4, containing 0.01 M E D T A and 0.3% BSA. After 5 days incubation at 4 °C, free labelled peptide was separated from antibody-bound peptide by charcoal precipitation. The assay was able to detect changes of I fmol per tube at 95% confidence. A serial dilution curve of human spinal cord extract was parallel to the standard curve of synthetic porcine neuromedin B in the NB 8 assay system (data are not shown). The neuromedin B-like immunoreactivities were characterized both by gel permeation and by reverse phase HPLC. Spinal cord extracts (0.5 ml) of dorsal regions were applied onto a Sephadex G-50 superfine column (0.9 × 60 cm) and eluted at a flow rate of 3.5 ml/h at 4 °C with the assay buffer, containing 0.2 M sodium chloride and 0.3% BSA. The column was precalibrated with dextran blue. horse heart cytochrome C and [125I]Nai. Synthetic porcine neuromedin B standard was chromatographed separately to determine the respective elution coefficients (Kav)L H P L C runs were performed on/~ Bondapak C-18 reverse phase columns (Waters) and eluted with a linear gradient from 25% to 40% acetonitrile containing 0.1% trifluoroacetic acid for 60 min. Prior to loading, original tissue extracts and pooled aliquots of each of the two main separated peaks of dorsal sacral cord from a previous G-50 column run. were purified on Sep-Pak C-18 reverse phase cartridges (Waters) and injected onto the column in 2 ml of 12% acetonitrile. The flow rate was 1 ml/min and 1-ml fractions were collected for subsequent radioimmunoassay. The rat liver extracts containing exogenous neuromedin B were also analysed by gel permeation and HPLC. All exogenous immunoreactwity after boiling coeluted with unboiled synthetic neuromedin B as a single peak.
200"
1:0 Kay Fig. 1. Gel permeation profiles of neuromedin B-like immunoreactivity in extracts of dorsal sacral region of human spinal cord on a Sephadex G-50 superfine column (0.9 × 60 cm): CC, horse heart cytochrome C; NB, synthetic porcine neuromedin B. Concentrations are expressed as fmol/fraction. K,,. isthe elution coefficientL The regional concentrations of neuromedm B-like immunoreactivity are shown in Table 1. The peptide concentrations were slightly higher in dorsal than ventral cord and increased caudally. The dorsal sacral regions show the highest level and the levels were significantly higher than the ventral sacral regions (P < 0.05. Student's paired t-test). The caudal accumulation of neuromedin B in the human spinal cord is a similar finding to that of many other neuroregulatory peptides 1.3,4. This has been attributed to a caudal decrease in the proportion of white matter, which has a lower peptide content I. Chromatographic analyses are shown in Figs. 1 and 2. Fig. 1 illustrates a representative gel permeation chromatographic profile for neuromedin B within the sacral region of the human dorsal cord. The prevalent molecular form coeluted with synthetic porcine neuromedin B (K~, = 0.87 _+ 0.01. n - 3) and comprised 9 0 - 9 5 % of total neuromedin B-like immunoreactivity. The apparently bigger molecular weight form. present in much lesser quantity, was eluted at around a K,,. of 0.60 indicating a moi. wt. in the range 2000-4000 dalton, depending on conformation All column recoveries were m excess of 70%. Fractionation of original tissue extracts of human spinal cord by H P L C also revealed the prevalent molecular form of the peptide in this tissue to coelute with the synthetic porcine neu-
185 fmol/fraction
NB
% acetonitrile
60-
30-
....
r----i]
A
20
£
,6
__._.------'-----''-10 -
B
100
20
......t40 o 10
20 30 40 50 retention time (rain)
60
70
Fig. 2. Reverse-phase high-pressure liquid chromatographic analyses of neuromedin B-like immunoreactivity in extracts of dorsal sacral region of human spinal cord and pooled aliquots of two major molecular forms eluted on a Sephadex G-50 superfine column. A: human dorsal sacral spinal cord. B: peak of larger molecular form rerun from G-50 column. C: peak of smaller molecular form rerun from G-50 column. NB, synthetic porcine neuromedin B. Concentrations are expressed as fmol/fraction. The dotted line shows the acetonitrile gradient.
romedin B (Fig. 2A). There were two other molecular forms in the spinal cord extract which eluted earlier and later than the major peak. However, the earlier peak has also appeared as a small subfraction of synthetic porcine neuromedin B standard and it is possible, therefore, that it is a degradative product. Separate HPLC fractionation of the two pooled aliquots of the major molecular forms after previous elution on gel permeation chromatography again revealed that the prevalent form eluted in the identical position to the porcine neuromedin B standard and the earlier gel eluting form was on HPLC the more hydrophobic form (Fig. 2B, C). Since it has been proposed that a proteolytic processing pathway of the related bombesin-like peptides from the 1-27 to the 18-27 form exists in mammalian brain 9, this larger neuromedin B molecular form might be similarly
processed. This study shows an approximately 10-fold greater concentration of neuromedin B in human spinal cord when compared to bombesin-like immunoreactivity in the same extracts~, the latter being detected with an antibody which cross-reacted fully with mammalian gastrin-releasing peptide and amphibian bombesin. It is of interest that in human gut extracts the ratio is reversed, there being very low concentrations of neuromedin B but considerable amounts of the decapeptide mammalian bombesin (gastrin-releasing peptide; unpublished observation). The significance of these differences is not apparent but, in spite of their structural similarities, the role of these peptides in the spinal cord may be quite different, especially as the active sequences have been apparently conserved from the time when amphibians and higher vertebrates separated. It is of interest that the amounts of neuromedin B in the cat cord are very similar to those in the human spinal cord, with the pig and rat spinal cord showing higher levels but of the same order of magnitude s . These interspecies quantitative differences are unlikely to be due to postmortem handling of tissues as the pig, rat and cat spinal cords were all dissected and extracted immediately after the animals were sacrificed while the human spinal cords were collected in a manner in which a number of peptides have been shown to be stable postmortem 2 and levels showed a similar dispersion and no correlation with time after death before extraction. The regional distribution of neuromedin B and its chromatographic characteristics are also strikingly similar in the 4 species so far studied. The possibility of undertaking immunocytochemical studies in freshly fixed animal cord showed neuromedin B-staining fibres predominantly within the substantia gelatinosa and around autonomic nuclei, suggesting a role in the modulation of sensory input and autonomic outflowS. Further studies will be required to confirm these points and demonstrate its precise physiological roles. This study was supported by grants from the British Diabetic Association and the Medical Research Council. M.N. acknowledges a British Council Scholarship (1983/84).
186 1 Anand, P., Gibson, S. J., McGregor, G. P., Blank, M. A., Ghatei, M. A., Bacarese-Hamilton, A. J., Polak, J. M. and Bloom, S. R., A VIP-containing system concentrated in the lumbosacral region of human spinal cord, Nature (Lond.), 305 (1983) 143-145. 2 Edwardson, J. A. and McDermott, J. R., Neurochemical pathology of brain peptide, Brit. reed. Bull., 38 (1982) 259-264. 3 Gibson, S. J., Polak, J. M., Allen, J. M., Adrian, T. E., Relly, J. S. and Bloom, S. R., The distribution and origin of a novel brain peptide, neuropeptide Y in the spinal cord of several mammals, J. cornp. Neurol., 227 (1984) 78-92. 4 Gibson, S. J., Polak, J. M., Bloom, S. R., Sabate, I. M., Mulderry, P. K., Ghatei, M. A., McGregor, G. P., Morrison, J. F. B., Kelly, J. S., Evans, R. M. and Rosenfeld, M. G., Calcitonin gene-related peptide (CGRP) immunoreactivity in spinal cord of eight other species, J. Neurosci., 4 (1984) 3101-3111. 5 Laurent, T. C. and Killander, J., A theory of gel filtration
6
7
8
9
and its experimental verification, J. Chromatogr., 14 (1964} 317-330. Minamino, N., Kangawa, K. and Matsuo, H , Neuromedm B: a novel bombesin-like peptide identified in porcine spinal cord, Biochem. biophys. Re~. Commun., !14 (1983) 541-548. Namba, M., Ghatei, M. A., Adrian, T. E., Bacarese-Hamilton, A. J., Mulderry, P. K. and Bloom, S. R., Effect of neuromedin B on gut hormone secretion in the rat, Biomed. Res., 5 (1984) 229-234. Namba, M., Ghatei, M. A., Gibson, S. J., Polak, J. M. and Bloom, S. R., Distribution and localization of neuromedin B-like immunoreactivity in pig, cat and rat spinal cord, Neuroscience, in press. Roth, K. A., Evans, C. J., Lorenz, R. G., Weber, E., Barchas, J. D. and Chang, J. K., Identification of gastrin releasing peptide-related substance in guinea pig and rat brain, Biochem. biophys. Res. Commun.. 112 (1983) 528-536.
183
BRE 21011
Distribution and chromatographic characterization of neuromedin B-like immunoreactivity in the human spinal cord M. NAMBA, M. A. GHATEI, P. ANAND and S. R. BLOOM Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS ( U. K.
(Accepted April 16th, 1985) Key words: neuromedin B-like immunoreactivity - - spinal cord - - human
The quantitative regional distribution of neuromedin B-like immunoreactivity in normal postmortem human spinal cord was studied by a specific radioimmunoassay. Neuromedin B-like immunoreactivity was found in highest concentration in the dorsal part of the sacral cord. Chromatographic analyses by gel permeation and reverse-phase high-pressure liquid chromatography revealed two major peaks of neuromedin B-like immunoreactivity and the prevalent molecular form, approx. 90% of the total immunoreactivity, was chromatographically identical to synthetic porcine neuromedin B.
N e u r o m e d i n B is a novel d e c a p e p t i d e isolated from porcine spinal cord which shows a striking sequence h o m o l o g y with m e m b e r s of bombesin-like peptide family, particularly the peptides of litorin/ranatensin subfamily, at the C-terminal heptapeptide6. N e u r o m e d i n B shows p o t e n t contractile activity on uterine muscle and stimulatory effects on the release of gastrointestinal and pancreatic regulatory peptides with a profile very similar to that of bombesin6,7. Since the p e p t i d e has potent biological actions, we studied the distribution of n e u r o m e d i n B-like immunoreactivity in human spinal cord. H u m a n spinal cord specimens were collected postmortem from 8 adults (6 males, 2 females; mean 60 year, range 4 8 - 8 3 year) with no evidence of nervous system pathology. All cadavers were stored at 4 °C within 2 h of death, and the spinal cords r e m o v e d within 24 h, a period generally established for stability of n e u r o p e p t i d e s in p o s t m o r t e m specimens 2. For radioimmunoassay, transverse slices (0.5 cm thick) from cervical, thoracic, lumbar and sacral regions were divided into ventral and dorsal regions by section through the central canal and stored in liquid nitrogen. N e u r o m e d i n B was extracted by boiling in 0.5 M acetic acid (10 ml per g wet wt. tissue) for 10
min. Duplicate 20~ul aliquots of each extract were assayed by a specific radioimmunoassay. In order to investigate the efficiency of the extraction, synthetic n e u r o m e d i n B standard was incubated with fresh rat liver h o m o g e n a t e s , for 1 h (n = 4). The mixtures were then boiled in 0.5 M acetic acid for 10 rain. The recovery of exogenous n e u r o m e d i n B-like immunoreactivity after boiling was 102 _+ 6%. The r a d i o i m m u n o a s s a y is described in full elsewhereS. The antiserum (NB 8) was raised in a rabbit to synthetic porcine n e u r o m e d i n B (Cambridge Research Biochemical, Cambridgeshire, U . K . , Lot PMG/22/GIy/3) conjugated to bovine serum albumin (BSA) with glutaraldehyde and used at a final dilution of 1:7000. This antibody cross-reacted 70% with litorin and 60% with ranatensin, but showed no significant cross-reaction with other known neuropeptides including bombesin and substance P. [Try°] neuromedin B (Lot PMG/22/Tyr/5, C a m b r i d g e Research Biochemical) was iodinated by the conventional chloramine-T oxidation. The iodinated product was then purified by high-pressure liquid chromatography ( H P L C ) using a C-18 reverse phase column (10 ~ m , 3.9 x 300 mm) and eluted in 30% acetonitrile in an aqueous solution of 0.1% trifluoroacetic
Correspondence: S. R. Bloom, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London WI2 0HS, U,K.
00(16-8993/85/$(13.3(I © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
184 TABLE
fmol/fraction
1
Regional concentrations of neuromedin B-h'ke immum~reactivttv in human spinal cord
CC
N J';
400
Results are e x p r e s s e d as m e a n + S . E . M . in p m o l / g wet wt.
Cervical Thoracic Lumbar Sacral
Ventral
Dorsal
1.9 2.0 3.9 3.3
2.0 3.6 4.7 12.4
+_ 0.7 + 0.8 + 0.9 _+_0.8
+ (t.4 _+ 1.2 _+ (1.8 ± 1.7
acid. The assay was carried out in 0.06 M phosphate buffer, p H 7.4, containing 0.01 M E D T A and 0.3% BSA. After 5 days incubation at 4 °C, free labelled peptide was separated from antibody-bound peptide by charcoal precipitation. The assay was able to detect changes of I fmol per tube at 95% confidence. A serial dilution curve of human spinal cord extract was parallel to the standard curve of synthetic porcine neuromedin B in the NB 8 assay system (data are not shown). The neuromedin B-like immunoreactivities were characterized both by gel permeation and by reverse phase HPLC. Spinal cord extracts (0.5 ml) of dorsal regions were applied onto a Sephadex G-50 superfine column (0.9 × 60 cm) and eluted at a flow rate of 3.5 ml/h at 4 °C with the assay buffer, containing 0.2 M sodium chloride and 0.3% BSA. The column was precalibrated with dextran blue. horse heart cytochrome C and [125I]Nai. Synthetic porcine neuromedin B standard was chromatographed separately to determine the respective elution coefficients (Kav)L H P L C runs were performed on/~ Bondapak C-18 reverse phase columns (Waters) and eluted with a linear gradient from 25% to 40% acetonitrile containing 0.1% trifluoroacetic acid for 60 min. Prior to loading, original tissue extracts and pooled aliquots of each of the two main separated peaks of dorsal sacral cord from a previous G-50 column run. were purified on Sep-Pak C-18 reverse phase cartridges (Waters) and injected onto the column in 2 ml of 12% acetonitrile. The flow rate was 1 ml/min and 1-ml fractions were collected for subsequent radioimmunoassay. The rat liver extracts containing exogenous neuromedin B were also analysed by gel permeation and HPLC. All exogenous immunoreactwity after boiling coeluted with unboiled synthetic neuromedin B as a single peak.
200"
1:0 Kay Fig. 1. Gel permeation profiles of neuromedin B-like immunoreactivity in extracts of dorsal sacral region of human spinal cord on a Sephadex G-50 superfine column (0.9 × 60 cm): CC, horse heart cytochrome C; NB, synthetic porcine neuromedin B. Concentrations are expressed as fmol/fraction. K,,. isthe elution coefficientL The regional concentrations of neuromedm B-like immunoreactivity are shown in Table 1. The peptide concentrations were slightly higher in dorsal than ventral cord and increased caudally. The dorsal sacral regions show the highest level and the levels were significantly higher than the ventral sacral regions (P < 0.05. Student's paired t-test). The caudal accumulation of neuromedin B in the human spinal cord is a similar finding to that of many other neuroregulatory peptides 1.3,4. This has been attributed to a caudal decrease in the proportion of white matter, which has a lower peptide content I. Chromatographic analyses are shown in Figs. 1 and 2. Fig. 1 illustrates a representative gel permeation chromatographic profile for neuromedin B within the sacral region of the human dorsal cord. The prevalent molecular form coeluted with synthetic porcine neuromedin B (K~, = 0.87 _+ 0.01. n - 3) and comprised 9 0 - 9 5 % of total neuromedin B-like immunoreactivity. The apparently bigger molecular weight form. present in much lesser quantity, was eluted at around a K,,. of 0.60 indicating a moi. wt. in the range 2000-4000 dalton, depending on conformation All column recoveries were m excess of 70%. Fractionation of original tissue extracts of human spinal cord by H P L C also revealed the prevalent molecular form of the peptide in this tissue to coelute with the synthetic porcine neu-
185 fmol/fraction
NB
% acetonitrile
60-
30-
....
r----i]
A
20
£
,6
__._.------'-----''-10 -
B
100
20
......t40 o 10
20 30 40 50 retention time (rain)
60
70
Fig. 2. Reverse-phase high-pressure liquid chromatographic analyses of neuromedin B-like immunoreactivity in extracts of dorsal sacral region of human spinal cord and pooled aliquots of two major molecular forms eluted on a Sephadex G-50 superfine column. A: human dorsal sacral spinal cord. B: peak of larger molecular form rerun from G-50 column. C: peak of smaller molecular form rerun from G-50 column. NB, synthetic porcine neuromedin B. Concentrations are expressed as fmol/fraction. The dotted line shows the acetonitrile gradient.
romedin B (Fig. 2A). There were two other molecular forms in the spinal cord extract which eluted earlier and later than the major peak. However, the earlier peak has also appeared as a small subfraction of synthetic porcine neuromedin B standard and it is possible, therefore, that it is a degradative product. Separate HPLC fractionation of the two pooled aliquots of the major molecular forms after previous elution on gel permeation chromatography again revealed that the prevalent form eluted in the identical position to the porcine neuromedin B standard and the earlier gel eluting form was on HPLC the more hydrophobic form (Fig. 2B, C). Since it has been proposed that a proteolytic processing pathway of the related bombesin-like peptides from the 1-27 to the 18-27 form exists in mammalian brain 9, this larger neuromedin B molecular form might be similarly
processed. This study shows an approximately 10-fold greater concentration of neuromedin B in human spinal cord when compared to bombesin-like immunoreactivity in the same extracts~, the latter being detected with an antibody which cross-reacted fully with mammalian gastrin-releasing peptide and amphibian bombesin. It is of interest that in human gut extracts the ratio is reversed, there being very low concentrations of neuromedin B but considerable amounts of the decapeptide mammalian bombesin (gastrin-releasing peptide; unpublished observation). The significance of these differences is not apparent but, in spite of their structural similarities, the role of these peptides in the spinal cord may be quite different, especially as the active sequences have been apparently conserved from the time when amphibians and higher vertebrates separated. It is of interest that the amounts of neuromedin B in the cat cord are very similar to those in the human spinal cord, with the pig and rat spinal cord showing higher levels but of the same order of magnitude s . These interspecies quantitative differences are unlikely to be due to postmortem handling of tissues as the pig, rat and cat spinal cords were all dissected and extracted immediately after the animals were sacrificed while the human spinal cords were collected in a manner in which a number of peptides have been shown to be stable postmortem 2 and levels showed a similar dispersion and no correlation with time after death before extraction. The regional distribution of neuromedin B and its chromatographic characteristics are also strikingly similar in the 4 species so far studied. The possibility of undertaking immunocytochemical studies in freshly fixed animal cord showed neuromedin B-staining fibres predominantly within the substantia gelatinosa and around autonomic nuclei, suggesting a role in the modulation of sensory input and autonomic outflowS. Further studies will be required to confirm these points and demonstrate its precise physiological roles. This study was supported by grants from the British Diabetic Association and the Medical Research Council. M.N. acknowledges a British Council Scholarship (1983/84).
186 1 Anand, P., Gibson, S. J., McGregor, G. P., Blank, M. A., Ghatei, M. A., Bacarese-Hamilton, A. J., Polak, J. M. and Bloom, S. R., A VIP-containing system concentrated in the lumbosacral region of human spinal cord, Nature (Lond.), 305 (1983) 143-145. 2 Edwardson, J. A. and McDermott, J. R., Neurochemical pathology of brain peptide, Brit. reed. Bull., 38 (1982) 259-264. 3 Gibson, S. J., Polak, J. M., Allen, J. M., Adrian, T. E., Relly, J. S. and Bloom, S. R., The distribution and origin of a novel brain peptide, neuropeptide Y in the spinal cord of several mammals, J. cornp. Neurol., 227 (1984) 78-92. 4 Gibson, S. J., Polak, J. M., Bloom, S. R., Sabate, I. M., Mulderry, P. K., Ghatei, M. A., McGregor, G. P., Morrison, J. F. B., Kelly, J. S., Evans, R. M. and Rosenfeld, M. G., Calcitonin gene-related peptide (CGRP) immunoreactivity in spinal cord of eight other species, J. Neurosci., 4 (1984) 3101-3111. 5 Laurent, T. C. and Killander, J., A theory of gel filtration
6
7
8
9
and its experimental verification, J. Chromatogr., 14 (1964} 317-330. Minamino, N., Kangawa, K. and Matsuo, H , Neuromedm B: a novel bombesin-like peptide identified in porcine spinal cord, Biochem. biophys. Re~. Commun., !14 (1983) 541-548. Namba, M., Ghatei, M. A., Adrian, T. E., Bacarese-Hamilton, A. J., Mulderry, P. K. and Bloom, S. R., Effect of neuromedin B on gut hormone secretion in the rat, Biomed. Res., 5 (1984) 229-234. Namba, M., Ghatei, M. A., Gibson, S. J., Polak, J. M. and Bloom, S. R., Distribution and localization of neuromedin B-like immunoreactivity in pig, cat and rat spinal cord, Neuroscience, in press. Roth, K. A., Evans, C. J., Lorenz, R. G., Weber, E., Barchas, J. D. and Chang, J. K., Identification of gastrin releasing peptide-related substance in guinea pig and rat brain, Biochem. biophys. Res. Commun.. 112 (1983) 528-536.