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Adrenorphin immunoreactivity in rat brain
Neuropeptides
ADRENORPHIN
5
517-520, 1985
IMMUNOREACTIVITY
IN RAT BRAIN
Atsuro Miyata, Kensaku Mizuno, Mayumi Honzawa*, Masaya Tohyama* and Hisayuki Matsuo Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki 889-16, Japan *)Department of Anatomy, Institute of Higher Nervous Activity, Osaka University School of Medicine, Nakanoshima, Osaka 530, Japan (reprint requests to H.M.) ABSTRACT the first C-terminally amidated form of opioid peptides Adrenorphin is isolated from human pheochromocytoma tumor and is considered to be generated out of proenkephalin A by unique processing. By the highly specific and sensitive utilizing the antiserum against radioimmunoassay(RIA) procedure with high performance liquid chromatoqraphy(HPLC1, adrenorphin, combined with its immunoreactive adrenorphin in rat brain was verified to be identical authentic peptide. It has been revealed that adrenorphin immunoreactivity from those distributes widely in rat brain but in the unique pattern distinct of other endoqenous opioid peptides. Note that immunoreactive adrenorphin was most concentrated in the olfactory bulb, and appreciably in the hypothalamus study has revealed that and striatum. Furthermore, immunohistochemical adrenorphin-immunoreactive in hypothalamic region of rat were structures localized in the neurones of the arcuate nucleus. In addition, adrenorphinregions of the immunoreactive fibre plexus was found in the various periventricular zone and parahypothalamus, such as median eminence, may have a unique ventricular nucleus. These indicate that adrenorphin physiological function. INTRODUCTION Adrenorphin, recently identified by our group in human pheochromocytoma tumor, is the first C-terminally amidated opioid peptide to be found in mammalian tissues(l). Existence of an identical peptide ( metorphamide ) in bovine brain has been reported independently by Weber et al.(2). In addition to its structural uniqueness, the potent opioid activity of adrenorphin, 15 times as high as Met-enkephalin in guinea pig ileum assay, suggests that this peptide may have a physiological role of its own. Furthermore, the sequence corresponding to adrenorphin is present within proenkephalin A and followed by a qlycine residue, serving for C-terminai amidation, as a nitrogen donor implying that adrenorphin is programmed to be processed out of the precursor. Interestingly, the qlycine residue is connected to the unique sequence of -Arq-Pro-, which has not so far been regarded as a typical processing signal, 517
unlike the precursors of the other known C-terminally amidated hormones, where the glycine residue is commonly followed by a well known processing signal of paired basic residues. In this context, the distribution of the peptide in regions in the brain will provide various information of significance necessary for understanding not only its physiological function, but also its processing features. Accordingly, by use of an antiserum specific for adrenorphin, we have examined regional distribution of adrenorphin in rat brain, and studied immunohistochemically, as the present paper describes. METHODS Radioimmunoassay was performed in the described procedure(3). The antiserum was obtained from a New Zealand white rabbit immunized with adrenorphin conjugated to bovine thyroglobulin by the carbodiimide technique. The antiserum is highly specific for adrenorphin and does not crossreact significantly with gther known opioid peptides, such as des-amide-adrenorphin, adrenorphinphin-Gly , BAM-12P ,Met-enk and PH-8P(dyxiorphin(l-81) _ Radioiodinated adrenorphin was prepared by the lactoperoxidase method. Bound and free ligands were separated by the use of polyethyleneglycol. Samples were routinely assayed in duplicate. At a final dilution of 1:50,000, half maximum inhibition by adrenorphin at 48 pg/tube was observed (ca. 49 fmol) and adrenorphin was detectable as low as 2 pg/tube in this RIA system(3). Immediately after decapitation of rats, the brains were dissected according to the method of Glowinski and Iversen (5). After weighing, tissues were boiled for 10 min in 1M acetic acid containing 20 mM HCl to inactivate intrinsic proteases. After cooling, they were homogenized with a Polytron mixer for 60 set in 10 volume (v/w) of the above solution. The homogenates were centrifuged at 16,000 rpm for 20 min. An aliquot of the supernatant was neutralized by adding an equal volume of 1.3 M Tris solution . The solution thus prepared was used as a sample solution for RIA. For identification of inununoreactive adrenorphin in rat brain, one rat brain (1.96 g) was extracted as described above, and submitted to acetoneprecipitation at a concentration of 66%. the supernatant thus obtained was The fraction evaporated and loaded on a Sep-pak C-18 cartridge (Waters). of 60% CH CN and 0.1% TFA was concentrated, eluted with a solution and then HPLC3 The used column and solvent system are submitted to reverse phase of each fraction was lyophilized and An aliquot described in Fig.1 legend. submitted to RIA for adrenorphin. intracardially with ice-cold Zamboni's fixative fluid, After perfusion immersed in the same the hypothalamus and adrenal gland of rats were removed, Immunohistochemical procedure are the method of fixative for 24 hours. indirect immunofluorescence as well as the described(7). RESULTS AND DISCUSSION The specificity against and sensitivity of the antiserum adrenorphin prepared above enabled us to determine its regional distribution in rat brain. of distribution of adrenorphin , immunoreactive Prior to the investigation species present in rat brain extracts was identified as follows. As seen in Fig.1, a major peak of adrenorphin , possessing more than 90% of the total immunoreactivity, identical with authentic emerged at the position
518
300
0
I
I
I
IO
20
Yl
Tlme(mln.1 1: Adrenorphin-sulfoxide, 2: Fig.1. Reverse phase HPLC of total rat brain. Adrenorphin. Column: 4.0x250 mm, TSK LS-410 ODS SIL (C-18, Toyosoda). Solvent system: A linear gradient elution from (A) to (B) (40 min). Flow rate: 2.0 ml/min. (A) 10% TFA:H20:CH3CN = 1:90:10, (B) 10% TFA:H20:CH3CN = 1:40:60 (V/V) peptide. Thus it was proven that immunoreactive adrenorphin in rat brain was regional derived only from authentic peptide. Table 1 summarizes the distribution of ir-adrenorphin and those of other opioid peptides in rat brain. The order of concentration of adrenorphin olfactory bulb > is, hypothalamus = striatum>hippocampus > midbrain/thalamus= medulla/pans >cerebellum> cortex. the highest concentration of adrenorphin was. Interestingly, observed in the olfactory bulb where other opioid peptides including PH-8P are negligible, suggesting a physiological role of adrenorphin in the olfactory bulb. In the other regions, the content of adrenorphin was rather low, As compared with generated from proenkephalin A by Met-enk-Arg6-Phe'(6), processing at paired basic residues. orre ation between the Moreover "g 3 distributional patterns of adrenorphin and Met-enk-Arg -Phe was not observed, implying 3s not that adrenorpgin generated from proenkephalin A at equal levels with Met-enk-Arg -Phe in each region of the brain. On the other hand, PH-8P is generated from proenkephalin B by processing at -Arg-Pro- in a manner similar and is structurally regarded to adrenorphin, as a counterpart of adrenorphin, suggesting the presence of a common processing system. However the distribution of PH-8P shows no correlation to that of adrenorphin(3). The content of PH-8P was comparable to that of a-neoendorphin in each region, and its distribution shows a close correlation to that of o-neoendorphin, which is also generated from proenkephalin B. In regional distribution, the close ;;:;;;;t$p;
,',9~~et~~~;;"~",g~~~~~7,ands~~~~~~~~~r~~~~ a~~~no~~~info~~dge~~~~~~~
biosynthetic mechanism of its own, including C-terminal by a specific amidation. The unique distributional distributional pattern of adrenorphin in rat brain together with its structural uniqueness suggests that adrenorphin serves as an opioid peptide with a specialized physiological function distinct from other opioid peptides. 519
Table 1. Regional distribgtion,of adrenrophin in rat brain (compared with Met-enk-Arg -Phe , PH-8P and a-neo-endorphin) adrenorphin
Olfactory
bulb
Met-enk-An&Phe7#
314 2 92
Hippocampus
21.4t7.1
Striatum
26.9
--__
Cortex
4.9?
Cerebellum
9.2t1.4
2.9 ?: 0.2
30.5i7.2
* 6.6
232 f 16
1.0
a-neo-endorphin’
PH-BP
36.1
f. 8
19.8
f 3.4
Cl.0
40.4t
7.6
44.6
54.3+-
9.1
37.2i7.1
14.2
+ 2.4
10.7
2.2f0.3
f 7.1
+ 0.7 < 1.0
Midbrain/thalamus
16.6t4.6
Medulla/pans
15.4
+ 5.6
123 t 14
12.5+
2.4
12.2
?: 1.3
Hypothalamus
24.2
+ 5.1
229 f 4
62.7+
6.9
75.7
f 1.7
Results
are
animals.
(
expressed l
:
Ref.4,
110+
as pmol/g #:
Ref.6.
wet
tissue.
9.1
All
25.7
values
are
+ 3.2
mean ? standard
17.8?
2.2
deviation
for
six
)
On the other hand, immunohistochemical study has revealed the presence of adrenorphin-immunoreactive cells in the arcuate nucleus and adrenorphin immunoreactive fiber plexus in the median eminence, suggesting the existence In addition, of a tubero-infundibular system of adrenorphin. we also demonstrated the existence of an adrenorphin-immunoreactive fiver plexus in the paraventricular nucleus, particularly in the parvocellular portion, implying that adrenorphin may relate to the extrahypohtalamic function. REFERENCE 1. Matsuo, H., Miyata, A, and Mizuno, K. (1983). Novel C-terminally opioid peptide in human pheochromocytoma. Nature 305: 721-723.
amidated
2. Weber, E., Esch, F.S., Bohlen, P., et al. (1983). Metorphamide: Isolation, structure, and biologic activity of an amidated opioid octapeptide from bovine brain. Proc. Natl. Acad. Sci. USA 80: 7362-7366. 3. Miyata, A., Mizuno, K., Minamino, N., and Matsuo, H. (1984). Regional distribution of adrenorphin in rat brain : comparative study with PH-8P. Biochem. Biophys. Res. Commun. 120: 1030-1036. 4. Minamino, N., Kitamura, K., Hayashi, Y. et al. (1981). Regional distribution of a-neo-endorphin in rat brain and pituitary. Biochem. Biophys. Res. Commun. 102: 226-234. 5. Glowinski, J. and Iversen, L.L. (1966). Regional studies of catecholamines in the rat brain-I. J. Neurochem. 13: 655-669. 6. Giraud, P., Castanas, E.,6Pate y,,G. et al. (1983). Regional distribution of methionine-enkephalin-Arg -Phe in the rat brain : Comparative study with the distribution of the other opioid peptides. J. Neurochem. 41: 154-160. 7. Coons, A.H.(1958). Fluorescent antibody methods. In: Danielli, J.F.(ed.) General cytochemical Methods. Academic Press, New York, p.399-422.
520
ADRENORPHIN
5
517-520, 1985
IMMUNOREACTIVITY
IN RAT BRAIN
Atsuro Miyata, Kensaku Mizuno, Mayumi Honzawa*, Masaya Tohyama* and Hisayuki Matsuo Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki 889-16, Japan *)Department of Anatomy, Institute of Higher Nervous Activity, Osaka University School of Medicine, Nakanoshima, Osaka 530, Japan (reprint requests to H.M.) ABSTRACT the first C-terminally amidated form of opioid peptides Adrenorphin is isolated from human pheochromocytoma tumor and is considered to be generated out of proenkephalin A by unique processing. By the highly specific and sensitive utilizing the antiserum against radioimmunoassay(RIA) procedure with high performance liquid chromatoqraphy(HPLC1, adrenorphin, combined with its immunoreactive adrenorphin in rat brain was verified to be identical authentic peptide. It has been revealed that adrenorphin immunoreactivity from those distributes widely in rat brain but in the unique pattern distinct of other endoqenous opioid peptides. Note that immunoreactive adrenorphin was most concentrated in the olfactory bulb, and appreciably in the hypothalamus study has revealed that and striatum. Furthermore, immunohistochemical adrenorphin-immunoreactive in hypothalamic region of rat were structures localized in the neurones of the arcuate nucleus. In addition, adrenorphinregions of the immunoreactive fibre plexus was found in the various periventricular zone and parahypothalamus, such as median eminence, may have a unique ventricular nucleus. These indicate that adrenorphin physiological function. INTRODUCTION Adrenorphin, recently identified by our group in human pheochromocytoma tumor, is the first C-terminally amidated opioid peptide to be found in mammalian tissues(l). Existence of an identical peptide ( metorphamide ) in bovine brain has been reported independently by Weber et al.(2). In addition to its structural uniqueness, the potent opioid activity of adrenorphin, 15 times as high as Met-enkephalin in guinea pig ileum assay, suggests that this peptide may have a physiological role of its own. Furthermore, the sequence corresponding to adrenorphin is present within proenkephalin A and followed by a qlycine residue, serving for C-terminai amidation, as a nitrogen donor implying that adrenorphin is programmed to be processed out of the precursor. Interestingly, the qlycine residue is connected to the unique sequence of -Arq-Pro-, which has not so far been regarded as a typical processing signal, 517
unlike the precursors of the other known C-terminally amidated hormones, where the glycine residue is commonly followed by a well known processing signal of paired basic residues. In this context, the distribution of the peptide in regions in the brain will provide various information of significance necessary for understanding not only its physiological function, but also its processing features. Accordingly, by use of an antiserum specific for adrenorphin, we have examined regional distribution of adrenorphin in rat brain, and studied immunohistochemically, as the present paper describes. METHODS Radioimmunoassay was performed in the described procedure(3). The antiserum was obtained from a New Zealand white rabbit immunized with adrenorphin conjugated to bovine thyroglobulin by the carbodiimide technique. The antiserum is highly specific for adrenorphin and does not crossreact significantly with gther known opioid peptides, such as des-amide-adrenorphin, adrenorphinphin-Gly , BAM-12P ,Met-enk and PH-8P(dyxiorphin(l-81) _ Radioiodinated adrenorphin was prepared by the lactoperoxidase method. Bound and free ligands were separated by the use of polyethyleneglycol. Samples were routinely assayed in duplicate. At a final dilution of 1:50,000, half maximum inhibition by adrenorphin at 48 pg/tube was observed (ca. 49 fmol) and adrenorphin was detectable as low as 2 pg/tube in this RIA system(3). Immediately after decapitation of rats, the brains were dissected according to the method of Glowinski and Iversen (5). After weighing, tissues were boiled for 10 min in 1M acetic acid containing 20 mM HCl to inactivate intrinsic proteases. After cooling, they were homogenized with a Polytron mixer for 60 set in 10 volume (v/w) of the above solution. The homogenates were centrifuged at 16,000 rpm for 20 min. An aliquot of the supernatant was neutralized by adding an equal volume of 1.3 M Tris solution . The solution thus prepared was used as a sample solution for RIA. For identification of inununoreactive adrenorphin in rat brain, one rat brain (1.96 g) was extracted as described above, and submitted to acetoneprecipitation at a concentration of 66%. the supernatant thus obtained was The fraction evaporated and loaded on a Sep-pak C-18 cartridge (Waters). of 60% CH CN and 0.1% TFA was concentrated, eluted with a solution and then HPLC3 The used column and solvent system are submitted to reverse phase of each fraction was lyophilized and An aliquot described in Fig.1 legend. submitted to RIA for adrenorphin. intracardially with ice-cold Zamboni's fixative fluid, After perfusion immersed in the same the hypothalamus and adrenal gland of rats were removed, Immunohistochemical procedure are the method of fixative for 24 hours. indirect immunofluorescence as well as the described(7). RESULTS AND DISCUSSION The specificity against and sensitivity of the antiserum adrenorphin prepared above enabled us to determine its regional distribution in rat brain. of distribution of adrenorphin , immunoreactive Prior to the investigation species present in rat brain extracts was identified as follows. As seen in Fig.1, a major peak of adrenorphin , possessing more than 90% of the total immunoreactivity, identical with authentic emerged at the position
518
300
0
I
I
I
IO
20
Yl
Tlme(mln.1 1: Adrenorphin-sulfoxide, 2: Fig.1. Reverse phase HPLC of total rat brain. Adrenorphin. Column: 4.0x250 mm, TSK LS-410 ODS SIL (C-18, Toyosoda). Solvent system: A linear gradient elution from (A) to (B) (40 min). Flow rate: 2.0 ml/min. (A) 10% TFA:H20:CH3CN = 1:90:10, (B) 10% TFA:H20:CH3CN = 1:40:60 (V/V) peptide. Thus it was proven that immunoreactive adrenorphin in rat brain was regional derived only from authentic peptide. Table 1 summarizes the distribution of ir-adrenorphin and those of other opioid peptides in rat brain. The order of concentration of adrenorphin olfactory bulb > is, hypothalamus = striatum>hippocampus > midbrain/thalamus= medulla/pans >cerebellum> cortex. the highest concentration of adrenorphin was. Interestingly, observed in the olfactory bulb where other opioid peptides including PH-8P are negligible, suggesting a physiological role of adrenorphin in the olfactory bulb. In the other regions, the content of adrenorphin was rather low, As compared with generated from proenkephalin A by Met-enk-Arg6-Phe'(6), processing at paired basic residues. orre ation between the Moreover "g 3 distributional patterns of adrenorphin and Met-enk-Arg -Phe was not observed, implying 3s not that adrenorpgin generated from proenkephalin A at equal levels with Met-enk-Arg -Phe in each region of the brain. On the other hand, PH-8P is generated from proenkephalin B by processing at -Arg-Pro- in a manner similar and is structurally regarded to adrenorphin, as a counterpart of adrenorphin, suggesting the presence of a common processing system. However the distribution of PH-8P shows no correlation to that of adrenorphin(3). The content of PH-8P was comparable to that of a-neoendorphin in each region, and its distribution shows a close correlation to that of o-neoendorphin, which is also generated from proenkephalin B. In regional distribution, the close ;;:;;;;t$p;
,',9~~et~~~;;"~",g~~~~~7,ands~~~~~~~~~r~~~~ a~~~no~~~info~~dge~~~~~~~
biosynthetic mechanism of its own, including C-terminal by a specific amidation. The unique distributional distributional pattern of adrenorphin in rat brain together with its structural uniqueness suggests that adrenorphin serves as an opioid peptide with a specialized physiological function distinct from other opioid peptides. 519
Table 1. Regional distribgtion,of adrenrophin in rat brain (compared with Met-enk-Arg -Phe , PH-8P and a-neo-endorphin) adrenorphin
Olfactory
bulb
Met-enk-An&Phe7#
314 2 92
Hippocampus
21.4t7.1
Striatum
26.9
--__
Cortex
4.9?
Cerebellum
9.2t1.4
2.9 ?: 0.2
30.5i7.2
* 6.6
232 f 16
1.0
a-neo-endorphin’
PH-BP
36.1
f. 8
19.8
f 3.4
Cl.0
40.4t
7.6
44.6
54.3+-
9.1
37.2i7.1
14.2
+ 2.4
10.7
2.2f0.3
f 7.1
+ 0.7 < 1.0
Midbrain/thalamus
16.6t4.6
Medulla/pans
15.4
+ 5.6
123 t 14
12.5+
2.4
12.2
?: 1.3
Hypothalamus
24.2
+ 5.1
229 f 4
62.7+
6.9
75.7
f 1.7
Results
are
animals.
(
expressed l
:
Ref.4,
110+
as pmol/g #:
Ref.6.
wet
tissue.
9.1
All
25.7
values
are
+ 3.2
mean ? standard
17.8?
2.2
deviation
for
six
)
On the other hand, immunohistochemical study has revealed the presence of adrenorphin-immunoreactive cells in the arcuate nucleus and adrenorphin immunoreactive fiber plexus in the median eminence, suggesting the existence In addition, of a tubero-infundibular system of adrenorphin. we also demonstrated the existence of an adrenorphin-immunoreactive fiver plexus in the paraventricular nucleus, particularly in the parvocellular portion, implying that adrenorphin may relate to the extrahypohtalamic function. REFERENCE 1. Matsuo, H., Miyata, A, and Mizuno, K. (1983). Novel C-terminally opioid peptide in human pheochromocytoma. Nature 305: 721-723.
amidated
2. Weber, E., Esch, F.S., Bohlen, P., et al. (1983). Metorphamide: Isolation, structure, and biologic activity of an amidated opioid octapeptide from bovine brain. Proc. Natl. Acad. Sci. USA 80: 7362-7366. 3. Miyata, A., Mizuno, K., Minamino, N., and Matsuo, H. (1984). Regional distribution of adrenorphin in rat brain : comparative study with PH-8P. Biochem. Biophys. Res. Commun. 120: 1030-1036. 4. Minamino, N., Kitamura, K., Hayashi, Y. et al. (1981). Regional distribution of a-neo-endorphin in rat brain and pituitary. Biochem. Biophys. Res. Commun. 102: 226-234. 5. Glowinski, J. and Iversen, L.L. (1966). Regional studies of catecholamines in the rat brain-I. J. Neurochem. 13: 655-669. 6. Giraud, P., Castanas, E.,6Pate y,,G. et al. (1983). Regional distribution of methionine-enkephalin-Arg -Phe in the rat brain : Comparative study with the distribution of the other opioid peptides. J. Neurochem. 41: 154-160. 7. Coons, A.H.(1958). Fluorescent antibody methods. In: Danielli, J.F.(ed.) General cytochemical Methods. Academic Press, New York, p.399-422.
520