- Email: [email protected]
Parallel distribution of immunoreactive α-neo-endorphin and dynorphin in rat and human tissue
Neuropeptides 2: 211-225, 1982
PARALLEL DISTRIBUTION OF IMMUNOREACTIVE a-NEO-ENDORPHIN DYNORPHIN IN RAT AND HUMAN TISSUE
AND
D. Maysingerl, V. Hijlltl, B.R. Seizingerl, P. Mehraein2, A. Pasi3 IDepartment of Neuropharmacology, Max-Planckand A. Herzl. Institut fiir Psychiatric, Kraepelinstrasse 2, D-8000 Miinchen 40; 2Department of Neuropathology, Medizinische Hochschule Hannover, D-3000 Hannover (G.F.R.); 3Gerichtlich Medizinisches Institut der Universitzt Ziirich, Ziirichbergstrasse 8, CH-8028 Ziirich 7 (Switzerland) (reprint requests to DM). ABSTRACT Using antibodies directed against a-neo-endorphin and dynorphin-(l-13) a striking parallelism was found between the regional distribution of the two opioid peptides in various rat and human tissues. In the rat, the highest levels were found in the neurointermediate pituitary, followed by those in the adenohypophysis, the hypothalamus and the striatum. High levels were also found in the spinal cord, whereas the cerebellum contained only small amounts of the immunoreactive peptides. In peripheral tissues, high concentrations of both were detected in the gut and surprisingly, the lungs, whereas other tissues such as the adrenal glands, the kidney and the liver did not contain measurable amounts. The distribution pattern in the human brain was similar to that in the rat. A particularly high concentration of both of the peptides was found in the substantia nigra. In contrast to the rat pituitary, the human pituitary contained only small amounts of the immunoreactive opioid peptides.In all tissues of both species, the levels of immunoreactive (ir-1 a-neo-endorphin were somewhat higher than those of ir-dynorphin with the ir-aneo-endorphin/ir-dynorphin ratio varying between 1 and 3. In response to dehydration, ir-a-neo-endorphin, ir-dynorphin, vasopressin and leu-enkephalin were depleted from the neurohypophysis of the rat indicating a similar regulation of the pools of these peptides in the neurohypophysis. A common biosynthetic origin of ir-a-neo-endorphin and vasopressin, is unlikely, since Brattleboro rats which do not synthesize vasopressin possess levels of ir-a-neo-endorphin in the neurohypophysis comparable to those of control rats. Gelfiltration and HPLC analysis of the immunoreactive components in the rat pituitary lobes revealed that the vast majority of the immunoreactivity in the neurointermediate pituitary coelutes with synthetic a-neo-endorphin, whereas a substantial part of that in the adenohypophysis appears to comprise a material with a molecular size of about boo0 daltons. There was no evidence for the existence of substantial amounts of I3-neo211
endorphin in the rat pituitary. The possibility is discussed that the close parallelism in the distribution and secretion of ir-aneo-endorphin and ir-dynorphin might indicate a common biosynthetic origin for these peptides. INTRODUCTION In 1979, a-neo-endorphin and dynorphin, two highly active opiate peptides, both containing the sequence of the opioid pentapeptide leucine-enkephalin at their N-terminus were isolated from the porcine hypothalamus and pituitary, and partially sequenced (1,2). Very recently, the complete primary sequences of the decapeptide a-neo-endorphin and the heptadecapeptide dynorphin have been elucidated (3,4,5). In addition, two N-terminal fragments of a-neo-endorphin and dynorphin have been isolated and sequenced: O-neo-endorphin (= a-neo-endorphin-(l-9)) and dynorphin-(l-8) (6,7). Further, it was recently demonstrated that dynorphin-(l-8) was the major dynorphin-related opioid peptide in the pituitary of rats (8). By the use of antibodies against dynorphin-(l-13) and a-neoendorphin the distribution of both peptides has been examined in brain and pituitaries of rats (9-12) and ir-dynorphin has also been determined in human brain and pituitary (13). The present study provides evidence for a striking parallelism in the regional distribution of a-neo-endorphin and dynorphin in various tissues of the rat, as well as in the human brain and pituitary. Moreover, the study demonstrates a concomitant regulation of the pituitary pools of immunoreactive a-neo-endorphin and dynorphin in response to sodium chloride dehydration. MATERIAL AND METHODS Male Sprague-Dawley rats weighing 200-220 g Animals. (Jautz, Kisslegg, FRG) and homozygous Brattleboro rats weighing 320-340 g (Central Proefdieren Bedrijf, Zeist, The Netherlands) were used. In dehydration experiments Sprague-Dawley rats were divided into two groups each consisting of 10 animals. The control group received tap water for five days: the experimental group received a 2% (w/v) NaCl solution. Food was given to both groups ad lib. The rats were decapitated between 2-4 p.m. Brains were quickly removed and dissected as described by Glowinsky and Iversen (14). Pituitaries were divided in situ into the anterior and neurointermediate lobe (= posterior lobe with adhering pars intermedia). Human brains and pituitaries were obtained from 4 male subjects of 18.29.57 and 69 vears. Death resulted from acute heart attack (2) and suicide (2): In each case there was no evidence of primary cerebral disease. Brains and pituitary glands were frozen at -7oOC between 3 and 6 hours after death and dissected after thawing at O°C under a stereomicroscope. Dissected brain samples 212
were then refrozen at -8OOC until extraction
of peptides.
Extraction procedure. Tissues from eight rat brain areas (cerebellum, pans/medulla, hypothalamus, thalamus, midbrain, hippocampus, striatum and cortex and 12 human brain areas (see table 2) were incubated in 2-4 v/w of 0.1 M HCl for 10 min. at 96 C. The tissues were homogenized and then centrifuged at 12000 g for 10 min. Supernatants were adjusted to pH 7.5 with 0.1 N NaOH and 50 ~1 aliquots assayed for ir-a-neo-endorphin and irdynorphin .by radioimmunoassay. 125* a-Neo-endorphin was iodinated with Na Iodination procedure. emnlovinc the chloramin-T method under conditions analogous to B-endorphin radiolabelling (15). The radiolabeled peptide was separated from iodide by chromatography on a P-4 column using 0.1 M acetic acid as a mobile phase. Development of antibodies for a-neo-endorphin ("Agathe"). a-Neo-endorphin (4.5 mg) and thyrogkobulin (25.0 mg) were dissolved in 4 ml of water and cooled to 0 C. 2.5 mg of carbodiimide dissolved in 0.5 ml of water was added to the above mixture and stirred overnight at O°C. After exhaustive dialysis against 0.9% NaCl the conjugated peptide was lyophilized. The conjugated peptide was emulsified with Freund's complete adjuvant (incomplete adjuvant after the first injection) and injected intradermally at multiple sites on the back of two New Zealand white rabbits. After an initial dose of 1.5 mg the rabbits were boostered at 3 week intervals with 0.5 mg immunogen each. 14 days after the last injection the animals were test bled, and the antiserum of rabbit "Agathe" which proved to have the highest titer and sensitivity was used for radioimmunological determination of ir-aneo-endorphin. Radioimmunoassay procedure. Aliquots of the same tissue extracts were assayed for ir-a-neo-endorphin and ir-dynorphin according to the protocol described previously (15). For ir-a-neo-endorphin determinations, the antiserum "Agathe" (see above) was used, and for ir-dynorphin antiserum "Lucia" (generous gift of Dr. A. Goldstein). It should be noted that dynorphin-(l-17) exhibits the same cross-reactivity as dynorphin- (I-13) to "Lucia", though this antiserum is directed against dynorphin-(l-13) (IO). In addition, the antiserum exhibits no substantial avidity for a-neoendorphin- (l-10) and B-neo-endorphin (less than 0.1% cross-reactivity). Assays were carried out in Eppendorf tubes (1.5 ml); the incubation mixture consisted of 50 ~1 neutralized tissue extract (pH 7.4) or standard solution, 100 ~1 of either antiserum "Agathe" (final dilution 1:9000) or dynorphiq241-13) antiserum "Lucia" (final dilution 1:10000), 50 ~1 of I-labeled ligand all diluted up to a final voluge of 0.5 ml “buffer D" (16). The mixture was equilibrated at 4 C for 14-16 hours; thereafter antibodybound peptides were separated from the unbound peptides by adding 500 bl of charcoal suspension followed by centrifugation (2 min at 3000 g). Radioactivity was measured in the supernatant. For details see Hijllt et al. (15). 213
Gelfiltration. 50 neurointermediate lobes and 50 anterior pituitary lobes were pooled in Eppendorf tubes and incubated with boiling in 1.2 ml of 0.1 M HCl. Tissugs were homogenized and centrifuged at 140 000 g for 45 min at 4 C. 1 ml aliquot of the supernatant was collected and applied to Sephadex G-50 superfine column (0.9 x 90 cm), and then eluted with 1 M acetic acid at a flow rate of 5 ml/h at 6 C. The column was calibrated with bovine serum albumin, porcine B-lipotropin, dynorphin-(l-8), dynorphin(l-17), a-neo-endorphin and 3H-met-enkephalin. HPLC was perHigh performance liquid chromatography (HPLC). formed with a fi-Bondapak C 18 reverse phase column (3.9 x 300 mm). The column was eluted with 1 M acetic acid (pH 2.5) with a linear gradient of acetonitrile from 5% to 40% within 35 min at a flow rate of 2 ml/min. 1 ml fractions were collected, lyophylized and assayed for ir-a-neo-endorphin. The recoveries for synthetic and endogenous ir-a-neo-endorphin and ir-dynorphin used on HPLC were about 90%. Porcine B-lipotropin was a generous gift from Dr. Substances. Laszlo Grbf, Budapest, Hungary; dynorphin-(l-8), dynorphin-(l-13), dynorphin-(l-17), a-neo-endorphin, B-neo-endorphin, leu-enkephalin, human I3-endorphin, BAM 22-P were from Peninsula, San Carlos, USA. Na1251 and 3H-met-enkephalin were from Amersham, Braunschweig, FRG (specific activity 15.6 mCi (579 MBq) 125I/c1g of iodine). RESULTS Figure 1 depicts the characteristics of the antiserum "Agathe" directed against a-neo-endorphin. At a final dilution of I:9000 the antiserum recognizes a- and 8-neo-endorphin with a high avidity, but does not recognizes dynorphin-related opioid peptides (dynorphin-(l-8), -(l-13), -(l-17)), an opioid peptide from bovine adrenal medulla (BAM-22 P), human R-endorphin or leucine-enkephalin. The detection limit of the antiserum was about IO-15 fmoles/tube. Concentrations of ir-a-neo-endorphin and ir-dynorphin simultaneously measured in the brain, pituitary and peripheral organs of rats are presented in Table 1. By far the highest concentration of ir-a-neo-endorphin was found in the neurointermediate pituitary. The concentrations in the anterior pituitary, although much lower than in the neurointermediate pituitary, are nevertheless higher than in any other tissue investigated. Ir-a-neo-endorphin and ir-dynorphin were also found in peripheral organs, particularly in the gastrointestinal tract. The parallel distribution of these peptides in various areas is expressed by the ratio ir-a-neo-endorphin/ir-dynorphin. While the absolute concentrations varied by more than IOOO-fold, the 214
pituitary
2.1 2.2 1.7
1.1 0.4 1.0 < <
1.0 1.0
< < <
Liver Kidney Adrenal
medulla
<
1.8 1.0
1.0
1.0
Tissues frm two groups of rats each consisting of 10 animals were extracted and each extract ws assayed for both ir-a-neo-endorphinand ir-dynorphin (see Mathods). Values represent mean 2 S.E.M. of 10 tissues. Ihe detection limits were: brain areas, 0.5 pies/g; the pituitary, 5 ml&g; and the peripheral tissues, 1.0 pnoles/g.
Lungs
2.2 -+ 5.9 -+
Colon
0.9
3.7
5.8 -+ 3.5 -+
1.0
2.2
2.1
6.9 12.9 -+ 7.5 -+
1.7
1.0
108.0 -+ 30.1 1140.1 + 250.7 110.1 -+ 27.4 1940.5 -+ 320.8
15.0 +
6.6
1.0 -+ 3.5 -+
Stomach
Duodenum-jejunum
Neurointermediate
Anterior
pituitary
23.9 +
Spinal cord
0.3
4.9 -+ 0.5 -+
2.2
1.6
1.2
2.5
5.5
1.5
3.1
1.1
1.5
10.5 -+ 7.1 -+
2.2
3.0
1.1 2.7
3.2
5.3
0.2
Cerebellum
Cortex
8.1 -+ 0.7 -+
Medulla/pans
3.1
5.7
2.4 10.2
Ratio
1.7
15.7 -+ 8.8 +
Hippocampus
Midbrain
7.9 -+ 10.1 +
4.7
8.1
-+ 10.5 +
16.8 39.2
Ir-dynorphin (pmol/g)
IN THE
1.1
23.7 + 22.7 -+
Thalamus
Striatum
-+ 33.6 +
94.1
AND IR-DYNORPHIN
OF THE RAT
Ir-a-neo-endorphin (pmol/g)
ORGANS
OF IR-a-NEO-ENDORPHIN
AND PERIPHERAL
DISTRIBUTION
BRAIN, PITUITARY
REGIONAL
Hypothalamus
Structure
Table 1.
a-neo-endwpt~&\
lbneo-endorphin
0-L
05
5
I
1
50
500
non-
5lloo
5olh
labeled peptide(fmolesltube)
Specificity of the a-neo-endorphin antiserum "Agathe". The ability of various synthetic opioid peptides to compete with 125-iodinated a-neo-endorphin for common binding sites has been tested. ir-a-neo-endorphin/ir-dynorphin
ratio did not exceed 3.2.
As demonstrated in figure 2A/B, the vast majority of ir-aneo-endorphin in the neurointermediate pituitary and a substantial amount of ir-a-neo-endorphin from the anterior pituitary comigrated with synthetic a-neo-endorphin on a Sephadex G-50 column. However, a considerable amount of the total a-neo-endorphinimmunoreactivity in the anterior pituitary is composed by a highmolecular weight species with an apparent molecular weight of ca. 8000 (Fig. 2A). Although the neurointermediate pituitary does not contain this molecular weight form, it contains small amounts of an about 15000 dalton peptide, cross-reacting with the a-neoendorphin antiserum (Fig. 2A). For further identification, the major immunoreactive species of the neurointermediate pituitary, comigrating with synthetic a-neo-endorphin on gelfiltration, were analzyed on a HPLC C 18 reverse phase column: More than 95% of the total a-neo-endorphinimmunoreactivity comigrated with synthetic a-neo-endorphin (Fig. 3) - It should be noted that no immunoreactivity could be detected in the position of ir-B-neo-endorphin, although this peptides would be well recognized by the ir-a-neo-endorphin-antiserum "Agathe" (see Fig. 1). This indicated that the u-neo-endorphin-
216
anterior pituitary
=
lo-
!,a
b
ll
1
neurointermediate pituitary VO a 11
b 1
c de Vt 111 1
.o ‘; g 84 i 6c E 0. b$ -0 f A 2cu a .L10
20
30
I 40
I 60
64
I 70
fraction number Fig. 2.
Separation of a-neo-endorphin immunoreactive peptides in rat pituitary lobes by Sephadex G-50. Tissue extracts equivalent to 40 rat anterior or neurointermediate lobes were subjected to gelfiltration on a Sephadex G-50 column and the fractions assayed as described in the text. Arrows indicate the elution volumes of: bovine serum albumin (void volume, Vo), porcine B-lipotropin (a); human B-endorphin (b); Dynorphin- (l-17) (c); dynorphin- (I-13) (d); a-neoendorphin (e); H-met-enkephalin (total volume, V,).
immunoreactivity in the neurointermediate pituitary consists almost exclusively of ir-a-neo-endorphin, while ir-B-neo-endorphin does not occur in substantial amounts. Levels of ir-dynorphin in the neurointermediate pituitary of rats were recently reported to be greatly depleted in response to sodium chloride imbibition, whereas levels of ir-dynorphin in the anterior pituitary remained unchanged (17,18). Figure 4 illustrates the effect of dehydration on pituitary levels of ir-a-neo-endorphin. In agreement with the data acquired with ir-dynorphin, a highly significant depletion of ir-a-neo-endorphin levels in the neurointermediate pituitary was observed though the levels in the anterior pituitary were not significantly changed. Pituitary levels of ir-a-neo-endorphin and ir-dynorphin were also determined in 3 homozygous Brattleboro rats, a rat strain unable to synthesize vasopressin. While no ir-vasopressin was detectable, the
217
I-
+
0
15
20
25
retention
Fig. 3.
time
( min I
Reverse phase HPLC separation of immunoreactive components of rat neurointermediate components comigrating with synthetic a-neo-endorphin on Sephadex G-50 column. Sample: 10% of pooled aliquots of the fractions 55-59 of the gelfiltration of the neurointermediate pituitaries (see Fig. 2). Column: 3.9 x 300 mm, b-Bondapack C 18 (Waters Assoc.); Solvent system: 1 M acetic acid (pH 2.5); linear gradient of acetonitrile 5-40%. Arrows indicate the retention times of: a-neo-endorphin (a); met-enkephalin (b); O-neo-endorphin (c); dynorphin(l-8) (d); leu-enkephalin (e); dynorphin-(l-17) (f).
Brattleboro rat had unaltered levels of ir-a-neo-endorphin and ir-dynorphin in comparison to those of Sprague-Dawely rats (200220 g) (data not shown). Table 2 provides data concerning the regional distribution of ir-a-neo-endorphin in comparison to ir-dynorphin in human brain and pituitary. Striking similarities can be observed in the distribution pattern; the highest concentrations were found in the substantia nigra and the anterior hypothalamus. Very low concentrations of both peptides were measurable in the cerebellum and, surprisingly, in the posterior pituitary. While the concentrations or ir-a-neo-endorphin and ir-dynorphin in the posterior (neurointermediate) pituitary of rats are almost IOO-fold higher than in most of the other tissues, the concentrations of these peptides in human posterior pituitary are even lower than
218
y (D
posterior
nigra
-+ -+ -+ -+
31.3 20.0 7.4 24.5
-+ -+
a.7
-+
3.8
12.2
-+
Pars nervosa
-+ 9.4
-+
1.4
3.1
0.5
0.7
1.2
1.8
-+ -+
4.2 1.7
7.4
6.1
-+
+
-+
-+
5.3
0.8
+
120.6
-+
-+
14.5 9.6
-+
-+
12.6 2.3
-+
1.9
1.3
0.1
0.6
0.9
1.3
30.1
1.0
3.2
0.9
3.4
6.3
-+ 20.1
1.1
1.6
24.1
-+
-+ 88.3
3.8
3.0
1.2
2.0
1.2
2.2
2.2
2.1
1.1
1.6
1.7
3.2
1.6
1.3
1.5
4.3
3.9
Brain regions frcm four and pituitariesfrom three human subjectswere extracted and each extract was assayValues represent mean -+ S.E.M. The detection ed for both ir-a-neo-endorphinand ir-dynorphin (see Methods). limit was 0.7 pm3les/g tissue.
Stalk
a.1
6.1
1.3
5.5
a.9
30.3
3.8
4.3
-+ 28.4 11.3
130.6
1.1
vermis
incl. lot. coeruleus
dorsal portion
-+
132.2
-+
-+
16.5
15.8
-+
12.0
Ratio
IN HUMAN BRAIN AND
Ir-dynorphin (pmol/g)
AND IR-DYNORPHIN
Ir-a-neo-endorphin (pmol/g)
OF IR-a-NEO-ENDORPHIN
Pars distalis
Pituitary
Cerebellum,
Medulla oblongata,
Pons, dorsal portion,
Substantia
gray matter
area
area
lateral nuclei
Periaqueductal
Putamen
Thalamus,
Thalamus, medial nuclei
anterior
Hypothalamus,
Hypothalamus,
Hippocampus
Cortex, frontal
DISTRIBUTION
lobe
PITUITARY
REGIONAL
Brain regions
Table 2.
neurointermediate pituitary
L
anterior pituitary
loo
_L
75
50
25
0
Fig. 4.
Effect of dehydration on immunoreactive a-neo-endorphin levels in neurointermediate and anterior pituitary lobes of rats. Rats received drinking water or 2% solution of. NaCl for 5 days. Columns indicate mean + S.E.M. (n=lO) *p
in the brain. The low levels of ir-dynorphin and ir-a-neo-endorphin in the human posterior pituitary are in good agreement with recent estimations of ir-dynorphin in human posterior pituitary (13).
DISCUSSION The present investigation demonstrates a close parallelism between the distribution of ir-a-neo-endorphin and ir-dynorphin in a variety of rat and human tissues. In rats, both of the peptides occur in highest concentrations in the neurointermediate pituitary followed by that in the
220
anterior pituitary, the hypothalamus and the striatum, whereas the cerebellum contains only very low concentrations of these peptides. The regional distributions of the immunoreactive peptides in the human brain were very similar to those found in the rat. In contrast to the rat pituitary, however, the human pituitary contains relatively low concentrations of ir-a-neo-endorphin and ir-dynorphin. This appears to reflect a species difference. Substantial post-mortem degradations of a-neo-endorphin and dynorphin in the human pituitary are unlikely, since the levels of other peptides such as B-endorphin and vasopressin in the human pituitary are similar to those in the rat pituitary (for discussion of this problem see (13)). Owing to the large size of the human brain, the concentrations of ir-a-neo-endorphin could be measured in a variety of brain structures. Of particular interest are the high concentrations of ir-a-neo-endorphin and ir-dynorphin in the substantia nigra indicating a putative functional relationship between the two peptides and the nigra-striatal dopaminergic system. In the rat, ir-a-neo-endorphin and ir-dynorphin were measured in several peripheral tissues. Relatively high concentrations of both peptides were found in the gastrointestinal tract, in particular in the jejunum. However, the peptides were not detectable in the kidney, the liver or the adrenal medulla. The lack of substantial amounts of these peptides in the adrenal medulla indicates that a-neo-endorphin and dynorphin are not biosynthetic intermediates in the processing of the common 50 k-daltons enkephalin precursor which contains in its aminoacid sequence seven copies of met-enkephalin and one copy of leu-enkephalin (19). On the other hand, significant amounts of ir-a-neo-endorphin and ir-dynorphin were found in the lungs. It is, as yet, unclear, into the large vascular whether the opioid peptides are absorbed bed of the lungs from the blood circulation or whether there are stored within neurons or cells intrinsic to the lung tissue. It is possible that the opioid peptides may play a functional role in modulating respiratory or secretory mechanisms by interacting with peripheral opioid receptors localized in the lungs, although such receptors have not, as yet, been demonstrated to exist by binding studies -in vitro. In addition to the parallelism of the distribution of ir-aneo-endorphin and ir-dynorphin, it seems that there are also common mechanisms for the regulation of the secretion of both peptides. Ir-a-neo-endorphin appears to be influenced by dehydration stimulus as indicated by the marked fall in the neurohypophyseal levels of ir-a-neo-endorphin after 5 days sodium chloride imbibition. The same pharmacological manipulation was previously shown to deplete concentrations of ir-vasopressin, ir-leu-enkephalin and ir-dynorphin (18,20). It seems unlikely that ir-a-neoendorphin derives from the same precursor molecule as vasopressin and the corresponding neurophysin (= pro-pressophysin (21)) since Brattleboro rats, which are unable to synthesize vasopressin, possess unchanged levels of ir-a-neo-endorphin in their neuro-
NEUR-
C
221
hypophysis (and also unchanged levels of ir-dynorphin and leu-enkephalin as reported previously (18)). In fact, the complete structure of pro-pressophysin elucidated by means of mRNA cloning, has not shown any aminoacid sequence related to a-neo-endorphin or dynorphin (D. Richter, personal communication). When the components in the rat pituitary lobes which react with the a-neo-endorphin antiserum were separated by gelfiltration, a further similarity between the distribution of immunoreactive a-neo-endorphin and dynorphin-like peptides became apparent. The neurointermediate pituitary contains predominantly immunoreactive peptides of smaller molecular weight such as ir-aneo-endorphin (present paper) or ir-dynorphin-(l-17) and ir-dynorphin-(l-8) (8,22). However, the anterior pituitary contains immunoreactive forms with higher molecular size. Ir-dynorphin in the anterior pituitary consists almost exclusively of a 6000 daltons peptide (22) and a substantial part of ir-a-neo-endorphin consists of a peptide with a molecular size of approximately 8000 daltons (see Fig. 2). This indicates that the enzymatic processing of a-neo-endorphin and dynorphin from putative precursor molecules is different in the anterior pituitary from that in the neurointermediate pituitary. An analoguous observation has been made for the common precursor for a-endorphin and ACTH (pro-opiomelanotropin (23)). This is predominantly processed into the larger peptides ACTH and R-lipotropin in the anterior pituitary, whereas in the intermediate pituitary a further processing into the smaller fragments, B-endorphin and a-MSH, occurs (24). Dynorphin-(l-17) appears to be further processed into dynorphin- (l-8) in the neurointermediate pituitary of rats (8). Therefore, a further processing of a-neo-endorphin into U-neo-endorphin (= a-neo-endorphin-(l-9) ), is possible. However, no evidence for the occurence of ir-I3-neo-endorphin in the rat neurointermediate pituitary was found, though this peptide if present, should have been detected by the antiserum used. Very recently, the regional distribution of ir-a-neo-endorphin the rat brain and pituitary has been reported by Minamino et al. (12). The order of ir-a-neo-endorphin concentrations found by these authors are very similar to those reported in the present paper. However, when comparing their data obtained for ir-a-neo-endorphin with that of the regional distribution of irdynorphin reported by Goldstein and Ghazarossian (IO), Minamino et al. (12) emphasized major differences between the relative and absolute concentrations of both immunoreactive peptides. In particular, they found that the hypothalamus contains about ten times more ir-a-neo-endorphin than ir-dynorphin. These differences, however, might be explained by differences in the dissection of the brain areas, and/or by variation in the method of extraction of the tissues and the different radioimmunoassay procedures used by the two groups. In contrast, when measuring both peptides in the same tissue extracts, the differences between the absolute concentrations of 222
ir-a-neo-endorphin and ir-dynorphin are much smaller. This is indicated by the ratio of 1 to 3 between ir-a-neo-endorphin to irdynorphin in a wide variety of tissues. The concentration ratios of met-enkephalin to leu-enkephalin in various brain regions show a greater variability (ratios from I:1 to 1:9 (2511, eventhough these peptides are believed to derive from a common precursor molecule (19,26). In view of the close parallelism in the distribution of ira-neo-endorphin and ir-dynorphin and of its concomitant secretion from the neurohypophysis, it appears possible that both opioid peptides may be present in a common neurone network or even originate from a common precursor molecule - a hypothesis which should be addressed by the use of biosynthesis studies. ACKNOWLEDGEMENTS The authors wish to thank M.J. Millan for stylistic revision of the English text. Assistance of the Alexander-von-HumboldtStiftung is gratefully acknowledged (D-M.). REFERENCES 1.
Kangawa, K., Matsuo, H. and Igarashi, M. (1979). a-Neoendorphin a big leu-enkephalin with potent opiate activity from porcine hypothalami. Biochem. Biophys. Res. Commun. 86: 153-160.
2.
Goldstein, A., Tachibana, S., Lowney, L-I., Hunkapillar, M. and Hood, L. (1979). Dynorphin-(I-131, an extraordinarily potent opioid peptide. Proc. Natl. Acad. Sci. (Wash.) 76: 6666-6670.
3.
Kangawa, K., Minamino, N., Chino, N., Sakakibara, S. and Matsuo, H. (1981). The complete amino-acid sequence of a-neoendorphin. Biochem. Biophys. Res. Commun. 99: 871-878.
4.
Tachibana, S., Araki, K., Ohya, S. and Yoshida, S. (1981). Isolation of dynorphin-like peptide from gut extract, P-20. Advances in Endogenous and Exogenous Opioids. International Narcotic Research Conference, July 26-30, Kyoto, Japan.
5.
Goldstein, A., Fischli, W., Lowney, L., Hunkapiller, M. and Hood, L. (1981). Porcine pituitary dynorphin: Complete acid sequence of the biologically active heptapeptide. Proc. Natl. Acad. Sci. USA, in press.
6.
Minamino, N., Kangawa, K., Chino, N., Matsuo, H. (1981). B-Neo-endorphin, a leu-enkephalin of porcine origin: its complete amino-acid sequence. Biochem. 99: 864-870.
7.
Minamino, N., Kangawa, K., Fukuda, A. and Matsuo, H. (1980). A new opioid octapeptide related to dynorphin from porcine hypothalamus. Biochem. Biophys. Res. Conunun. 95: 1475-1481.
223
Sakakibara, S. and new hypothalamic "big" purification and the Biophys. Res. Commun.
8.
Seizinger, B., Hollt, V. and Herz, A. (1981). Evidence for the occurence of the opioid octapeptide dynorphin-(l-8) in the neurointermediate pituitary of rats. Biochem. Biophys. Res. Commun. 102: 197-205.
9.
Hdllt, V., Haarmann, I., Bovermann, K., Jerlicz, M. and Herz, A. (1980). Dynorphin-related immunoreactive peptides in rat brain and pituitary. Neurosci. Lett. 18: 149-153.
10.
Goldstein, A. and Ghazarossian, V.E. (1980). Immunoreactive dynorphin in pituitary and brain. Proc. Natl. Acad. Sci. USA 77: 6207-6210.
11.
Millan, M.J., Tsang, Y.F., Przewsocki, Herz, A. (1981). The influence of foot brain, pituitary and spinal cord pools dynorphin in rats. Neurosci. Lett. 24:
12.
Minamino, N., Kitamura, K., Hayashi, Y., Kangawa, K. and Matsuo, H. (1981). Regional distribution of a-neo-endorphin in rat brain and pituitary. Biochem. Biophys. Res. Commun. 102: 226-234.
13.
Gramsch, C., Hbllt, V., Pasi, A., Mehraein, P. and Herz, A. (7982). Immunoreactive dynorphin in human brain and pituitary. Brain Res., in press.
14.
Glowinsky, J. and Iversen, L. (1966). Regional studies of catecholamines in the rat brain. I. Disposition of 3H-norepinephrine, 3H-dopamine and 3H-DOPA in various regions of the brain. J. Neurochem. 13: 655-669.
15.
Hollt, V., Przew&ocki, R. and Herz, A. (1978). Radioimmunoassay of B-endorphin. Basal and stimulated levels in extracted rat plasma. Naunyn-Schmiedeberg's Arch. Pharmacol. 303: 171-174.
16.
Guillemin, R., Ling, N. and Vargo, Th. (1977). Radioinununoassay for a-endorphin and B-endorphin. Biochem. Biophys. Res. Commun. 77: 361-366.
17.
Hollt, V., Haarmann, I., Seizinger, B. and Herz, A. (1980). Sodium chloride imbibition decreases levels of immunoreactive dynorphin in the neurointermediate pituitary of rats. Europ. J. Pharmacol. 68: 239-240.
18.
Hijllt, V., Haarmann, I., Seizinger, B.R. and Herz, A. (1981). Levels of dynorphin-(l-13) immunoreactivity in rat neurointermediate pituitaries are concomitantly altered with those of leucine-enkephalin and vasopressin in response to various endocrine manipulations. Neuroendocrinology 33: 333-339.
19.
Lewis, R.V., Stern, A.S., Kimura, S., Rossier, J., Stein, S., and Udenfriend, S. (1980). An about 50000-dalton protein in adrenal medulla: A common precursor of Met- and Leu-enkephalin. Science 208: 1459-1461.
20.
Rossier, J., Battenberg, E., Pittman, Q., Bayon, A., Koda, L., Miller, R., Guillemin, R. and Bloom, F. (1979). Hypothalamic enkephalin neurones may regulate the neurohypophysis. Nature (London) 277: 653-655. 224
R., Hollt, V. and shock stress upon of immunoreactive 75-79.
21.
Russel, J.T., Brownstein, M.J. and Gainer, H. (1979). Trypsin liberates an arginine vasopressin-like peptide and neurophysin from a Mr 20000 putative common precursor. Proc. Natl. Acad. Sci. USA 76: 6086-6090.
22.
Seizinger, B-R., Hiillt, V. and Herz, A. (1981). Immunoreactive dynorphin in the rat adenohypophysis consists exclusively of 6000 dalton species. Biochem. Biophys. Res. Commun. 103: 256-263.
23.
Crine, P., Gossard, F., Seidah, N.G., Blanchette, L., Lis, M. and Chretien, M. (1979). Concomitant synthesis of B-endorphin and a-melanotropin from two forms of pro-opiomelanocortin in the rat pars intermedia. Proc. Natl. Acad. Sci. USA 76: 5085-5089.
24.
Gramsch, C., Kleber, G., Hijllt, V., Pasi,'A., Mehraein, P. and Herz, A. (1980). Pro-opiocortin fragments in human and rat brain: B-endorphin and a-MSH are the predominant peptides. Brain Res. 192: 109-119.
25.
Smith, T.W., Hughes, J., Kosterlitz, H.W. and Sosa, R.P. (1976). Enkephalins: isolation, distribution and function. In: Kosterlitz, H.W. (ed.) Opiates and Endogenous Opioid Peptides. North-Holland Biomedical Press, Amsterdam, p. 57-62.
26.
Beaumont, A., Fuentes, J.A. and Metters, K.M. (1980). Opioid peptides precursor in striatum. FEBS Letters 122: 135-137.
Accepted 10 March 1982
225
PARALLEL DISTRIBUTION OF IMMUNOREACTIVE a-NEO-ENDORPHIN DYNORPHIN IN RAT AND HUMAN TISSUE
AND
D. Maysingerl, V. Hijlltl, B.R. Seizingerl, P. Mehraein2, A. Pasi3 IDepartment of Neuropharmacology, Max-Planckand A. Herzl. Institut fiir Psychiatric, Kraepelinstrasse 2, D-8000 Miinchen 40; 2Department of Neuropathology, Medizinische Hochschule Hannover, D-3000 Hannover (G.F.R.); 3Gerichtlich Medizinisches Institut der Universitzt Ziirich, Ziirichbergstrasse 8, CH-8028 Ziirich 7 (Switzerland) (reprint requests to DM). ABSTRACT Using antibodies directed against a-neo-endorphin and dynorphin-(l-13) a striking parallelism was found between the regional distribution of the two opioid peptides in various rat and human tissues. In the rat, the highest levels were found in the neurointermediate pituitary, followed by those in the adenohypophysis, the hypothalamus and the striatum. High levels were also found in the spinal cord, whereas the cerebellum contained only small amounts of the immunoreactive peptides. In peripheral tissues, high concentrations of both were detected in the gut and surprisingly, the lungs, whereas other tissues such as the adrenal glands, the kidney and the liver did not contain measurable amounts. The distribution pattern in the human brain was similar to that in the rat. A particularly high concentration of both of the peptides was found in the substantia nigra. In contrast to the rat pituitary, the human pituitary contained only small amounts of the immunoreactive opioid peptides.In all tissues of both species, the levels of immunoreactive (ir-1 a-neo-endorphin were somewhat higher than those of ir-dynorphin with the ir-aneo-endorphin/ir-dynorphin ratio varying between 1 and 3. In response to dehydration, ir-a-neo-endorphin, ir-dynorphin, vasopressin and leu-enkephalin were depleted from the neurohypophysis of the rat indicating a similar regulation of the pools of these peptides in the neurohypophysis. A common biosynthetic origin of ir-a-neo-endorphin and vasopressin, is unlikely, since Brattleboro rats which do not synthesize vasopressin possess levels of ir-a-neo-endorphin in the neurohypophysis comparable to those of control rats. Gelfiltration and HPLC analysis of the immunoreactive components in the rat pituitary lobes revealed that the vast majority of the immunoreactivity in the neurointermediate pituitary coelutes with synthetic a-neo-endorphin, whereas a substantial part of that in the adenohypophysis appears to comprise a material with a molecular size of about boo0 daltons. There was no evidence for the existence of substantial amounts of I3-neo211
endorphin in the rat pituitary. The possibility is discussed that the close parallelism in the distribution and secretion of ir-aneo-endorphin and ir-dynorphin might indicate a common biosynthetic origin for these peptides. INTRODUCTION In 1979, a-neo-endorphin and dynorphin, two highly active opiate peptides, both containing the sequence of the opioid pentapeptide leucine-enkephalin at their N-terminus were isolated from the porcine hypothalamus and pituitary, and partially sequenced (1,2). Very recently, the complete primary sequences of the decapeptide a-neo-endorphin and the heptadecapeptide dynorphin have been elucidated (3,4,5). In addition, two N-terminal fragments of a-neo-endorphin and dynorphin have been isolated and sequenced: O-neo-endorphin (= a-neo-endorphin-(l-9)) and dynorphin-(l-8) (6,7). Further, it was recently demonstrated that dynorphin-(l-8) was the major dynorphin-related opioid peptide in the pituitary of rats (8). By the use of antibodies against dynorphin-(l-13) and a-neoendorphin the distribution of both peptides has been examined in brain and pituitaries of rats (9-12) and ir-dynorphin has also been determined in human brain and pituitary (13). The present study provides evidence for a striking parallelism in the regional distribution of a-neo-endorphin and dynorphin in various tissues of the rat, as well as in the human brain and pituitary. Moreover, the study demonstrates a concomitant regulation of the pituitary pools of immunoreactive a-neo-endorphin and dynorphin in response to sodium chloride dehydration. MATERIAL AND METHODS Male Sprague-Dawley rats weighing 200-220 g Animals. (Jautz, Kisslegg, FRG) and homozygous Brattleboro rats weighing 320-340 g (Central Proefdieren Bedrijf, Zeist, The Netherlands) were used. In dehydration experiments Sprague-Dawley rats were divided into two groups each consisting of 10 animals. The control group received tap water for five days: the experimental group received a 2% (w/v) NaCl solution. Food was given to both groups ad lib. The rats were decapitated between 2-4 p.m. Brains were quickly removed and dissected as described by Glowinsky and Iversen (14). Pituitaries were divided in situ into the anterior and neurointermediate lobe (= posterior lobe with adhering pars intermedia). Human brains and pituitaries were obtained from 4 male subjects of 18.29.57 and 69 vears. Death resulted from acute heart attack (2) and suicide (2): In each case there was no evidence of primary cerebral disease. Brains and pituitary glands were frozen at -7oOC between 3 and 6 hours after death and dissected after thawing at O°C under a stereomicroscope. Dissected brain samples 212
were then refrozen at -8OOC until extraction
of peptides.
Extraction procedure. Tissues from eight rat brain areas (cerebellum, pans/medulla, hypothalamus, thalamus, midbrain, hippocampus, striatum and cortex and 12 human brain areas (see table 2) were incubated in 2-4 v/w of 0.1 M HCl for 10 min. at 96 C. The tissues were homogenized and then centrifuged at 12000 g for 10 min. Supernatants were adjusted to pH 7.5 with 0.1 N NaOH and 50 ~1 aliquots assayed for ir-a-neo-endorphin and irdynorphin .by radioimmunoassay. 125* a-Neo-endorphin was iodinated with Na Iodination procedure. emnlovinc the chloramin-T method under conditions analogous to B-endorphin radiolabelling (15). The radiolabeled peptide was separated from iodide by chromatography on a P-4 column using 0.1 M acetic acid as a mobile phase. Development of antibodies for a-neo-endorphin ("Agathe"). a-Neo-endorphin (4.5 mg) and thyrogkobulin (25.0 mg) were dissolved in 4 ml of water and cooled to 0 C. 2.5 mg of carbodiimide dissolved in 0.5 ml of water was added to the above mixture and stirred overnight at O°C. After exhaustive dialysis against 0.9% NaCl the conjugated peptide was lyophilized. The conjugated peptide was emulsified with Freund's complete adjuvant (incomplete adjuvant after the first injection) and injected intradermally at multiple sites on the back of two New Zealand white rabbits. After an initial dose of 1.5 mg the rabbits were boostered at 3 week intervals with 0.5 mg immunogen each. 14 days after the last injection the animals were test bled, and the antiserum of rabbit "Agathe" which proved to have the highest titer and sensitivity was used for radioimmunological determination of ir-aneo-endorphin. Radioimmunoassay procedure. Aliquots of the same tissue extracts were assayed for ir-a-neo-endorphin and ir-dynorphin according to the protocol described previously (15). For ir-a-neo-endorphin determinations, the antiserum "Agathe" (see above) was used, and for ir-dynorphin antiserum "Lucia" (generous gift of Dr. A. Goldstein). It should be noted that dynorphin-(l-17) exhibits the same cross-reactivity as dynorphin- (I-13) to "Lucia", though this antiserum is directed against dynorphin-(l-13) (IO). In addition, the antiserum exhibits no substantial avidity for a-neoendorphin- (l-10) and B-neo-endorphin (less than 0.1% cross-reactivity). Assays were carried out in Eppendorf tubes (1.5 ml); the incubation mixture consisted of 50 ~1 neutralized tissue extract (pH 7.4) or standard solution, 100 ~1 of either antiserum "Agathe" (final dilution 1:9000) or dynorphiq241-13) antiserum "Lucia" (final dilution 1:10000), 50 ~1 of I-labeled ligand all diluted up to a final voluge of 0.5 ml “buffer D" (16). The mixture was equilibrated at 4 C for 14-16 hours; thereafter antibodybound peptides were separated from the unbound peptides by adding 500 bl of charcoal suspension followed by centrifugation (2 min at 3000 g). Radioactivity was measured in the supernatant. For details see Hijllt et al. (15). 213
Gelfiltration. 50 neurointermediate lobes and 50 anterior pituitary lobes were pooled in Eppendorf tubes and incubated with boiling in 1.2 ml of 0.1 M HCl. Tissugs were homogenized and centrifuged at 140 000 g for 45 min at 4 C. 1 ml aliquot of the supernatant was collected and applied to Sephadex G-50 superfine column (0.9 x 90 cm), and then eluted with 1 M acetic acid at a flow rate of 5 ml/h at 6 C. The column was calibrated with bovine serum albumin, porcine B-lipotropin, dynorphin-(l-8), dynorphin(l-17), a-neo-endorphin and 3H-met-enkephalin. HPLC was perHigh performance liquid chromatography (HPLC). formed with a fi-Bondapak C 18 reverse phase column (3.9 x 300 mm). The column was eluted with 1 M acetic acid (pH 2.5) with a linear gradient of acetonitrile from 5% to 40% within 35 min at a flow rate of 2 ml/min. 1 ml fractions were collected, lyophylized and assayed for ir-a-neo-endorphin. The recoveries for synthetic and endogenous ir-a-neo-endorphin and ir-dynorphin used on HPLC were about 90%. Porcine B-lipotropin was a generous gift from Dr. Substances. Laszlo Grbf, Budapest, Hungary; dynorphin-(l-8), dynorphin-(l-13), dynorphin-(l-17), a-neo-endorphin, B-neo-endorphin, leu-enkephalin, human I3-endorphin, BAM 22-P were from Peninsula, San Carlos, USA. Na1251 and 3H-met-enkephalin were from Amersham, Braunschweig, FRG (specific activity 15.6 mCi (579 MBq) 125I/c1g of iodine). RESULTS Figure 1 depicts the characteristics of the antiserum "Agathe" directed against a-neo-endorphin. At a final dilution of I:9000 the antiserum recognizes a- and 8-neo-endorphin with a high avidity, but does not recognizes dynorphin-related opioid peptides (dynorphin-(l-8), -(l-13), -(l-17)), an opioid peptide from bovine adrenal medulla (BAM-22 P), human R-endorphin or leucine-enkephalin. The detection limit of the antiserum was about IO-15 fmoles/tube. Concentrations of ir-a-neo-endorphin and ir-dynorphin simultaneously measured in the brain, pituitary and peripheral organs of rats are presented in Table 1. By far the highest concentration of ir-a-neo-endorphin was found in the neurointermediate pituitary. The concentrations in the anterior pituitary, although much lower than in the neurointermediate pituitary, are nevertheless higher than in any other tissue investigated. Ir-a-neo-endorphin and ir-dynorphin were also found in peripheral organs, particularly in the gastrointestinal tract. The parallel distribution of these peptides in various areas is expressed by the ratio ir-a-neo-endorphin/ir-dynorphin. While the absolute concentrations varied by more than IOOO-fold, the 214
pituitary
2.1 2.2 1.7
1.1 0.4 1.0 < <
1.0 1.0
< < <
Liver Kidney Adrenal
medulla
<
1.8 1.0
1.0
1.0
Tissues frm two groups of rats each consisting of 10 animals were extracted and each extract ws assayed for both ir-a-neo-endorphinand ir-dynorphin (see Mathods). Values represent mean 2 S.E.M. of 10 tissues. Ihe detection limits were: brain areas, 0.5 pies/g; the pituitary, 5 ml&g; and the peripheral tissues, 1.0 pnoles/g.
Lungs
2.2 -+ 5.9 -+
Colon
0.9
3.7
5.8 -+ 3.5 -+
1.0
2.2
2.1
6.9 12.9 -+ 7.5 -+
1.7
1.0
108.0 -+ 30.1 1140.1 + 250.7 110.1 -+ 27.4 1940.5 -+ 320.8
15.0 +
6.6
1.0 -+ 3.5 -+
Stomach
Duodenum-jejunum
Neurointermediate
Anterior
pituitary
23.9 +
Spinal cord
0.3
4.9 -+ 0.5 -+
2.2
1.6
1.2
2.5
5.5
1.5
3.1
1.1
1.5
10.5 -+ 7.1 -+
2.2
3.0
1.1 2.7
3.2
5.3
0.2
Cerebellum
Cortex
8.1 -+ 0.7 -+
Medulla/pans
3.1
5.7
2.4 10.2
Ratio
1.7
15.7 -+ 8.8 +
Hippocampus
Midbrain
7.9 -+ 10.1 +
4.7
8.1
-+ 10.5 +
16.8 39.2
Ir-dynorphin (pmol/g)
IN THE
1.1
23.7 + 22.7 -+
Thalamus
Striatum
-+ 33.6 +
94.1
AND IR-DYNORPHIN
OF THE RAT
Ir-a-neo-endorphin (pmol/g)
ORGANS
OF IR-a-NEO-ENDORPHIN
AND PERIPHERAL
DISTRIBUTION
BRAIN, PITUITARY
REGIONAL
Hypothalamus
Structure
Table 1.
a-neo-endwpt~&\
lbneo-endorphin
0-L
05
5
I
1
50
500
non-
5lloo
5olh
labeled peptide(fmolesltube)
Specificity of the a-neo-endorphin antiserum "Agathe". The ability of various synthetic opioid peptides to compete with 125-iodinated a-neo-endorphin for common binding sites has been tested. ir-a-neo-endorphin/ir-dynorphin
ratio did not exceed 3.2.
As demonstrated in figure 2A/B, the vast majority of ir-aneo-endorphin in the neurointermediate pituitary and a substantial amount of ir-a-neo-endorphin from the anterior pituitary comigrated with synthetic a-neo-endorphin on a Sephadex G-50 column. However, a considerable amount of the total a-neo-endorphinimmunoreactivity in the anterior pituitary is composed by a highmolecular weight species with an apparent molecular weight of ca. 8000 (Fig. 2A). Although the neurointermediate pituitary does not contain this molecular weight form, it contains small amounts of an about 15000 dalton peptide, cross-reacting with the a-neoendorphin antiserum (Fig. 2A). For further identification, the major immunoreactive species of the neurointermediate pituitary, comigrating with synthetic a-neo-endorphin on gelfiltration, were analzyed on a HPLC C 18 reverse phase column: More than 95% of the total a-neo-endorphinimmunoreactivity comigrated with synthetic a-neo-endorphin (Fig. 3) - It should be noted that no immunoreactivity could be detected in the position of ir-B-neo-endorphin, although this peptides would be well recognized by the ir-a-neo-endorphin-antiserum "Agathe" (see Fig. 1). This indicated that the u-neo-endorphin-
216
anterior pituitary
=
lo-
!,a
b
ll
1
neurointermediate pituitary VO a 11
b 1
c de Vt 111 1
.o ‘; g 84 i 6c E 0. b$ -0 f A 2cu a .L10
20
30
I 40
I 60
64
I 70
fraction number Fig. 2.
Separation of a-neo-endorphin immunoreactive peptides in rat pituitary lobes by Sephadex G-50. Tissue extracts equivalent to 40 rat anterior or neurointermediate lobes were subjected to gelfiltration on a Sephadex G-50 column and the fractions assayed as described in the text. Arrows indicate the elution volumes of: bovine serum albumin (void volume, Vo), porcine B-lipotropin (a); human B-endorphin (b); Dynorphin- (l-17) (c); dynorphin- (I-13) (d); a-neoendorphin (e); H-met-enkephalin (total volume, V,).
immunoreactivity in the neurointermediate pituitary consists almost exclusively of ir-a-neo-endorphin, while ir-B-neo-endorphin does not occur in substantial amounts. Levels of ir-dynorphin in the neurointermediate pituitary of rats were recently reported to be greatly depleted in response to sodium chloride imbibition, whereas levels of ir-dynorphin in the anterior pituitary remained unchanged (17,18). Figure 4 illustrates the effect of dehydration on pituitary levels of ir-a-neo-endorphin. In agreement with the data acquired with ir-dynorphin, a highly significant depletion of ir-a-neo-endorphin levels in the neurointermediate pituitary was observed though the levels in the anterior pituitary were not significantly changed. Pituitary levels of ir-a-neo-endorphin and ir-dynorphin were also determined in 3 homozygous Brattleboro rats, a rat strain unable to synthesize vasopressin. While no ir-vasopressin was detectable, the
217
I-
+
0
15
20
25
retention
Fig. 3.
time
( min I
Reverse phase HPLC separation of immunoreactive components of rat neurointermediate components comigrating with synthetic a-neo-endorphin on Sephadex G-50 column. Sample: 10% of pooled aliquots of the fractions 55-59 of the gelfiltration of the neurointermediate pituitaries (see Fig. 2). Column: 3.9 x 300 mm, b-Bondapack C 18 (Waters Assoc.); Solvent system: 1 M acetic acid (pH 2.5); linear gradient of acetonitrile 5-40%. Arrows indicate the retention times of: a-neo-endorphin (a); met-enkephalin (b); O-neo-endorphin (c); dynorphin(l-8) (d); leu-enkephalin (e); dynorphin-(l-17) (f).
Brattleboro rat had unaltered levels of ir-a-neo-endorphin and ir-dynorphin in comparison to those of Sprague-Dawely rats (200220 g) (data not shown). Table 2 provides data concerning the regional distribution of ir-a-neo-endorphin in comparison to ir-dynorphin in human brain and pituitary. Striking similarities can be observed in the distribution pattern; the highest concentrations were found in the substantia nigra and the anterior hypothalamus. Very low concentrations of both peptides were measurable in the cerebellum and, surprisingly, in the posterior pituitary. While the concentrations or ir-a-neo-endorphin and ir-dynorphin in the posterior (neurointermediate) pituitary of rats are almost IOO-fold higher than in most of the other tissues, the concentrations of these peptides in human posterior pituitary are even lower than
218
y (D
posterior
nigra
-+ -+ -+ -+
31.3 20.0 7.4 24.5
-+ -+
a.7
-+
3.8
12.2
-+
Pars nervosa
-+ 9.4
-+
1.4
3.1
0.5
0.7
1.2
1.8
-+ -+
4.2 1.7
7.4
6.1
-+
+
-+
-+
5.3
0.8
+
120.6
-+
-+
14.5 9.6
-+
-+
12.6 2.3
-+
1.9
1.3
0.1
0.6
0.9
1.3
30.1
1.0
3.2
0.9
3.4
6.3
-+ 20.1
1.1
1.6
24.1
-+
-+ 88.3
3.8
3.0
1.2
2.0
1.2
2.2
2.2
2.1
1.1
1.6
1.7
3.2
1.6
1.3
1.5
4.3
3.9
Brain regions frcm four and pituitariesfrom three human subjectswere extracted and each extract was assayValues represent mean -+ S.E.M. The detection ed for both ir-a-neo-endorphinand ir-dynorphin (see Methods). limit was 0.7 pm3les/g tissue.
Stalk
a.1
6.1
1.3
5.5
a.9
30.3
3.8
4.3
-+ 28.4 11.3
130.6
1.1
vermis
incl. lot. coeruleus
dorsal portion
-+
132.2
-+
-+
16.5
15.8
-+
12.0
Ratio
IN HUMAN BRAIN AND
Ir-dynorphin (pmol/g)
AND IR-DYNORPHIN
Ir-a-neo-endorphin (pmol/g)
OF IR-a-NEO-ENDORPHIN
Pars distalis
Pituitary
Cerebellum,
Medulla oblongata,
Pons, dorsal portion,
Substantia
gray matter
area
area
lateral nuclei
Periaqueductal
Putamen
Thalamus,
Thalamus, medial nuclei
anterior
Hypothalamus,
Hypothalamus,
Hippocampus
Cortex, frontal
DISTRIBUTION
lobe
PITUITARY
REGIONAL
Brain regions
Table 2.
neurointermediate pituitary
L
anterior pituitary
loo
_L
75
50
25
0
Fig. 4.
Effect of dehydration on immunoreactive a-neo-endorphin levels in neurointermediate and anterior pituitary lobes of rats. Rats received drinking water or 2% solution of. NaCl for 5 days. Columns indicate mean + S.E.M. (n=lO) *p
in the brain. The low levels of ir-dynorphin and ir-a-neo-endorphin in the human posterior pituitary are in good agreement with recent estimations of ir-dynorphin in human posterior pituitary (13).
DISCUSSION The present investigation demonstrates a close parallelism between the distribution of ir-a-neo-endorphin and ir-dynorphin in a variety of rat and human tissues. In rats, both of the peptides occur in highest concentrations in the neurointermediate pituitary followed by that in the
220
anterior pituitary, the hypothalamus and the striatum, whereas the cerebellum contains only very low concentrations of these peptides. The regional distributions of the immunoreactive peptides in the human brain were very similar to those found in the rat. In contrast to the rat pituitary, however, the human pituitary contains relatively low concentrations of ir-a-neo-endorphin and ir-dynorphin. This appears to reflect a species difference. Substantial post-mortem degradations of a-neo-endorphin and dynorphin in the human pituitary are unlikely, since the levels of other peptides such as B-endorphin and vasopressin in the human pituitary are similar to those in the rat pituitary (for discussion of this problem see (13)). Owing to the large size of the human brain, the concentrations of ir-a-neo-endorphin could be measured in a variety of brain structures. Of particular interest are the high concentrations of ir-a-neo-endorphin and ir-dynorphin in the substantia nigra indicating a putative functional relationship between the two peptides and the nigra-striatal dopaminergic system. In the rat, ir-a-neo-endorphin and ir-dynorphin were measured in several peripheral tissues. Relatively high concentrations of both peptides were found in the gastrointestinal tract, in particular in the jejunum. However, the peptides were not detectable in the kidney, the liver or the adrenal medulla. The lack of substantial amounts of these peptides in the adrenal medulla indicates that a-neo-endorphin and dynorphin are not biosynthetic intermediates in the processing of the common 50 k-daltons enkephalin precursor which contains in its aminoacid sequence seven copies of met-enkephalin and one copy of leu-enkephalin (19). On the other hand, significant amounts of ir-a-neo-endorphin and ir-dynorphin were found in the lungs. It is, as yet, unclear, into the large vascular whether the opioid peptides are absorbed bed of the lungs from the blood circulation or whether there are stored within neurons or cells intrinsic to the lung tissue. It is possible that the opioid peptides may play a functional role in modulating respiratory or secretory mechanisms by interacting with peripheral opioid receptors localized in the lungs, although such receptors have not, as yet, been demonstrated to exist by binding studies -in vitro. In addition to the parallelism of the distribution of ir-aneo-endorphin and ir-dynorphin, it seems that there are also common mechanisms for the regulation of the secretion of both peptides. Ir-a-neo-endorphin appears to be influenced by dehydration stimulus as indicated by the marked fall in the neurohypophyseal levels of ir-a-neo-endorphin after 5 days sodium chloride imbibition. The same pharmacological manipulation was previously shown to deplete concentrations of ir-vasopressin, ir-leu-enkephalin and ir-dynorphin (18,20). It seems unlikely that ir-a-neoendorphin derives from the same precursor molecule as vasopressin and the corresponding neurophysin (= pro-pressophysin (21)) since Brattleboro rats, which are unable to synthesize vasopressin, possess unchanged levels of ir-a-neo-endorphin in their neuro-
NEUR-
C
221
hypophysis (and also unchanged levels of ir-dynorphin and leu-enkephalin as reported previously (18)). In fact, the complete structure of pro-pressophysin elucidated by means of mRNA cloning, has not shown any aminoacid sequence related to a-neo-endorphin or dynorphin (D. Richter, personal communication). When the components in the rat pituitary lobes which react with the a-neo-endorphin antiserum were separated by gelfiltration, a further similarity between the distribution of immunoreactive a-neo-endorphin and dynorphin-like peptides became apparent. The neurointermediate pituitary contains predominantly immunoreactive peptides of smaller molecular weight such as ir-aneo-endorphin (present paper) or ir-dynorphin-(l-17) and ir-dynorphin-(l-8) (8,22). However, the anterior pituitary contains immunoreactive forms with higher molecular size. Ir-dynorphin in the anterior pituitary consists almost exclusively of a 6000 daltons peptide (22) and a substantial part of ir-a-neo-endorphin consists of a peptide with a molecular size of approximately 8000 daltons (see Fig. 2). This indicates that the enzymatic processing of a-neo-endorphin and dynorphin from putative precursor molecules is different in the anterior pituitary from that in the neurointermediate pituitary. An analoguous observation has been made for the common precursor for a-endorphin and ACTH (pro-opiomelanotropin (23)). This is predominantly processed into the larger peptides ACTH and R-lipotropin in the anterior pituitary, whereas in the intermediate pituitary a further processing into the smaller fragments, B-endorphin and a-MSH, occurs (24). Dynorphin-(l-17) appears to be further processed into dynorphin- (l-8) in the neurointermediate pituitary of rats (8). Therefore, a further processing of a-neo-endorphin into U-neo-endorphin (= a-neo-endorphin-(l-9) ), is possible. However, no evidence for the occurence of ir-I3-neo-endorphin in the rat neurointermediate pituitary was found, though this peptide if present, should have been detected by the antiserum used. Very recently, the regional distribution of ir-a-neo-endorphin the rat brain and pituitary has been reported by Minamino et al. (12). The order of ir-a-neo-endorphin concentrations found by these authors are very similar to those reported in the present paper. However, when comparing their data obtained for ir-a-neo-endorphin with that of the regional distribution of irdynorphin reported by Goldstein and Ghazarossian (IO), Minamino et al. (12) emphasized major differences between the relative and absolute concentrations of both immunoreactive peptides. In particular, they found that the hypothalamus contains about ten times more ir-a-neo-endorphin than ir-dynorphin. These differences, however, might be explained by differences in the dissection of the brain areas, and/or by variation in the method of extraction of the tissues and the different radioimmunoassay procedures used by the two groups. In contrast, when measuring both peptides in the same tissue extracts, the differences between the absolute concentrations of 222
ir-a-neo-endorphin and ir-dynorphin are much smaller. This is indicated by the ratio of 1 to 3 between ir-a-neo-endorphin to irdynorphin in a wide variety of tissues. The concentration ratios of met-enkephalin to leu-enkephalin in various brain regions show a greater variability (ratios from I:1 to 1:9 (2511, eventhough these peptides are believed to derive from a common precursor molecule (19,26). In view of the close parallelism in the distribution of ira-neo-endorphin and ir-dynorphin and of its concomitant secretion from the neurohypophysis, it appears possible that both opioid peptides may be present in a common neurone network or even originate from a common precursor molecule - a hypothesis which should be addressed by the use of biosynthesis studies. ACKNOWLEDGEMENTS The authors wish to thank M.J. Millan for stylistic revision of the English text. Assistance of the Alexander-von-HumboldtStiftung is gratefully acknowledged (D-M.). REFERENCES 1.
Kangawa, K., Matsuo, H. and Igarashi, M. (1979). a-Neoendorphin a big leu-enkephalin with potent opiate activity from porcine hypothalami. Biochem. Biophys. Res. Commun. 86: 153-160.
2.
Goldstein, A., Tachibana, S., Lowney, L-I., Hunkapillar, M. and Hood, L. (1979). Dynorphin-(I-131, an extraordinarily potent opioid peptide. Proc. Natl. Acad. Sci. (Wash.) 76: 6666-6670.
3.
Kangawa, K., Minamino, N., Chino, N., Sakakibara, S. and Matsuo, H. (1981). The complete amino-acid sequence of a-neoendorphin. Biochem. Biophys. Res. Commun. 99: 871-878.
4.
Tachibana, S., Araki, K., Ohya, S. and Yoshida, S. (1981). Isolation of dynorphin-like peptide from gut extract, P-20. Advances in Endogenous and Exogenous Opioids. International Narcotic Research Conference, July 26-30, Kyoto, Japan.
5.
Goldstein, A., Fischli, W., Lowney, L., Hunkapiller, M. and Hood, L. (1981). Porcine pituitary dynorphin: Complete acid sequence of the biologically active heptapeptide. Proc. Natl. Acad. Sci. USA, in press.
6.
Minamino, N., Kangawa, K., Chino, N., Matsuo, H. (1981). B-Neo-endorphin, a leu-enkephalin of porcine origin: its complete amino-acid sequence. Biochem. 99: 864-870.
7.
Minamino, N., Kangawa, K., Fukuda, A. and Matsuo, H. (1980). A new opioid octapeptide related to dynorphin from porcine hypothalamus. Biochem. Biophys. Res. Conunun. 95: 1475-1481.
223
Sakakibara, S. and new hypothalamic "big" purification and the Biophys. Res. Commun.
8.
Seizinger, B., Hollt, V. and Herz, A. (1981). Evidence for the occurence of the opioid octapeptide dynorphin-(l-8) in the neurointermediate pituitary of rats. Biochem. Biophys. Res. Commun. 102: 197-205.
9.
Hdllt, V., Haarmann, I., Bovermann, K., Jerlicz, M. and Herz, A. (1980). Dynorphin-related immunoreactive peptides in rat brain and pituitary. Neurosci. Lett. 18: 149-153.
10.
Goldstein, A. and Ghazarossian, V.E. (1980). Immunoreactive dynorphin in pituitary and brain. Proc. Natl. Acad. Sci. USA 77: 6207-6210.
11.
Millan, M.J., Tsang, Y.F., Przewsocki, Herz, A. (1981). The influence of foot brain, pituitary and spinal cord pools dynorphin in rats. Neurosci. Lett. 24:
12.
Minamino, N., Kitamura, K., Hayashi, Y., Kangawa, K. and Matsuo, H. (1981). Regional distribution of a-neo-endorphin in rat brain and pituitary. Biochem. Biophys. Res. Commun. 102: 226-234.
13.
Gramsch, C., Hbllt, V., Pasi, A., Mehraein, P. and Herz, A. (7982). Immunoreactive dynorphin in human brain and pituitary. Brain Res., in press.
14.
Glowinsky, J. and Iversen, L. (1966). Regional studies of catecholamines in the rat brain. I. Disposition of 3H-norepinephrine, 3H-dopamine and 3H-DOPA in various regions of the brain. J. Neurochem. 13: 655-669.
15.
Hollt, V., Przew&ocki, R. and Herz, A. (1978). Radioimmunoassay of B-endorphin. Basal and stimulated levels in extracted rat plasma. Naunyn-Schmiedeberg's Arch. Pharmacol. 303: 171-174.
16.
Guillemin, R., Ling, N. and Vargo, Th. (1977). Radioinununoassay for a-endorphin and B-endorphin. Biochem. Biophys. Res. Commun. 77: 361-366.
17.
Hollt, V., Haarmann, I., Seizinger, B. and Herz, A. (1980). Sodium chloride imbibition decreases levels of immunoreactive dynorphin in the neurointermediate pituitary of rats. Europ. J. Pharmacol. 68: 239-240.
18.
Hijllt, V., Haarmann, I., Seizinger, B.R. and Herz, A. (1981). Levels of dynorphin-(l-13) immunoreactivity in rat neurointermediate pituitaries are concomitantly altered with those of leucine-enkephalin and vasopressin in response to various endocrine manipulations. Neuroendocrinology 33: 333-339.
19.
Lewis, R.V., Stern, A.S., Kimura, S., Rossier, J., Stein, S., and Udenfriend, S. (1980). An about 50000-dalton protein in adrenal medulla: A common precursor of Met- and Leu-enkephalin. Science 208: 1459-1461.
20.
Rossier, J., Battenberg, E., Pittman, Q., Bayon, A., Koda, L., Miller, R., Guillemin, R. and Bloom, F. (1979). Hypothalamic enkephalin neurones may regulate the neurohypophysis. Nature (London) 277: 653-655. 224
R., Hollt, V. and shock stress upon of immunoreactive 75-79.
21.
Russel, J.T., Brownstein, M.J. and Gainer, H. (1979). Trypsin liberates an arginine vasopressin-like peptide and neurophysin from a Mr 20000 putative common precursor. Proc. Natl. Acad. Sci. USA 76: 6086-6090.
22.
Seizinger, B-R., Hiillt, V. and Herz, A. (1981). Immunoreactive dynorphin in the rat adenohypophysis consists exclusively of 6000 dalton species. Biochem. Biophys. Res. Commun. 103: 256-263.
23.
Crine, P., Gossard, F., Seidah, N.G., Blanchette, L., Lis, M. and Chretien, M. (1979). Concomitant synthesis of B-endorphin and a-melanotropin from two forms of pro-opiomelanocortin in the rat pars intermedia. Proc. Natl. Acad. Sci. USA 76: 5085-5089.
24.
Gramsch, C., Kleber, G., Hijllt, V., Pasi,'A., Mehraein, P. and Herz, A. (1980). Pro-opiocortin fragments in human and rat brain: B-endorphin and a-MSH are the predominant peptides. Brain Res. 192: 109-119.
25.
Smith, T.W., Hughes, J., Kosterlitz, H.W. and Sosa, R.P. (1976). Enkephalins: isolation, distribution and function. In: Kosterlitz, H.W. (ed.) Opiates and Endogenous Opioid Peptides. North-Holland Biomedical Press, Amsterdam, p. 57-62.
26.
Beaumont, A., Fuentes, J.A. and Metters, K.M. (1980). Opioid peptides precursor in striatum. FEBS Letters 122: 135-137.
Accepted 10 March 1982
225