- Email: [email protected]
Distribution of immunoreactive dynorphin A1–8 in discrete nuclei of the rat brain: Comparison with dynorphin A
Brain Research, 307 (1984) 61-68 Elsevier
61
BILE 10222
Distribution of Immunoreactive Dynorphin AI_.8 in Discrete Nuclei of the Rat Brain: Comparison with Dynorphin A NADAV ZAMIR, MIKLOS PALKOVITS and MICHAEL J. BROWNSTEIN Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20205 (U.S.A.)
(Accepted January 3rd, 1984) Key words: dynorphin AI_8 - dynorphln A - - Leu-enkephalin - - RIA - - rat brain nuclei - - posterior pituitary
The distribution of immunoreactive (ir)-dynorphin A1..s (Dyn A1..s) in 78 microdissected rat brain areas as well as in the neurointermediate lobe of pituitary gland was determined using a highly specific radioimmunoassay. The highest concentrations of Dyn At..a in brain were found in substantia nigra (673.8 fmol/mg protein) and lateral preoptic area (565.1 fmol/mgprotein). High concentrations of ir-Dyn A1..s (> 240 fmol/mg protein) were found in 5 nuclei: ventral premamillary nucleus,:anterior hypothalamic nucleus, dorsomedial nucleus, areuate nucleus, and medullary reticular nuclei. Moderate concentrations of the peptide (between 120 and 240 fmol/mg protein) were found in 55 brain nuclei such as septal and amygdaloid nuclei, most diencephalic structures, mesencephalic nuclei, loons and medulla oblongata nuclei and others. Low concentrations of ir-Dyn A1..s (< 120 frnoFmgprotein) were found in 16 regions, e.g. frontal cortex, hlppocampus, caudate-putamen cortical amygdaloid nucleus, several thaiamic nuclei, mamillary body superior and inferior colliculi, cerebellar nuclei and others. The posterior thalamic nucleus has the lowest ir-Dyn AI_s concentration (62.0 fmol/mg protein). The neurointermediate lobe of the pituitary gland is extremely rich in ir-Dyn AI..a (4063.0 fmol/mg protein).
INTRODUCTION Dynorphin A1-8 (Dyn A1-8) is an endogenous opioid peptide which is an amino terminal fragment of dynorphin (Dyn A)4, 5,14,19. The structural relationship of the two peptides is illustrated below:
(a) dynorphin A: Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro1 5 8 -Lys-Leu-Lys-Trp-Asp--Asn-Gln 17 (b) dynorphin Al-s: Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile 1 5 8 It contains the amino acid sequence of Leu-enkeph-
alin at its amino terminus, and is a potent opiate agonist in the in vitro guinea pig myenteric plexus longitudinal muscle bioassay. It appears to be a highly selective ligand for the kappa opiate binding site2, 3. Dyn A and Dyn AI_8 are parts of the same precursor that gives rise to the neo-endorphinsS,9,15 and dynorphin B n. The amino acid sequence of this precursor has been deduced from the structure of D N A complementary to pro-dynorphin m R N A from porcine hypothalamus 7. Prodynorphin contains three Leuenkephalin sequences that form the N-termini of a-neo-endorphin, Dyn A, and Dyn B, respectively7. a-Neo-endorphin and the two dynorphins are flanked on either side by basic residues which serve as processing signals. These basic residues seem to be cleaved in preference to bases within a-neo-endorphin and the dynorphins. The latter can be cleaved, however. Presumably the A r g - P r o bond in Dyn A is severed and the Arg removed by carboxypeptidase B
Correspondence: N. Zamir, Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20205, U.S.A.
62 to yield Dyn AI_ s. Furthermore, it seems likely that Leu-enkephalin can also be liberated from Dyn A. Immunohistochemical studies have shown that perikarya, nerve fibers and terminals containing Dyn A are widely distributed throughout the central nervous system10.21,23. The distribution of Dyn AI_S, measured by RIA, in gross brain areas has been reported 25. The hypothalamus, striatum and midbrain are regions rich in the peptide; the hippocampus, medulla oblongata, and cortex contain moderate amounts of the peptide. The cerebellum has very little Dyn AI_8 (ref. 25). In this paper we describe the topographical distribution of ir-Dyn AI_s among 78 microdissected brain areas as well as neurointermediate lobe of the pituitary gland. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (Zivic-MiUer Laboratories, Allison Park, PA), weighing 220-250 g were housed under alternate 12-h periods of dark and light (lights on from 06.00 to 18.00 h) and were given standard rat chow and tap water ad libitum.
Tissue preparation and extraction The animals were killed by decapitation between 08.00 and 10.00 h. The brains were quickly removed and frozen on dry-ice. Brain areas were removed from 300/zm thick frozen coronal sections cut in a cryostat at --10 °C t6. Tissue from each microdissected area was pooled from two animals. Dyn AI_s and Dyn A were assayed in the same tissue samples. The distribution of the latter has been reported earlier 28. The pituitary gland was immediately removed and dissected into neurointermediate and anterior lobes. Tissue samples were placed in 1.5 ml conical Eppendorf tubes containing 200/xl of 0.1 N HC1 and transferred to a boiling water bath for 10 min. The tissue samples were then homogenized by sonication and 20/A aliquots of the homogenates were removed for protein determination t3. The extracts were centrifuged at 2000 g for 10 min at 4 °C, The supernatants were transferred to 12 x 75 mm polypropylene tubes and evaporated to dryness in a vacuum centrifuge.
Radioimmunoassay and specificity of the antiserum Samples were rehydrated in phosphate-buffered saline (pH 7.6) containing 0.1% gelatin, 0.1% bovine serum albumin, 0.1% Triton X-100 and 0.01% merthiolate. The antiserum was used at a final dilution of 1:120,000, which resulted in binding of 30-45% trace. Each sample was incubated at 4 °C in a 500/A volume that contained 300/A of sample in assay buffer, 100 ktl of [125I]labeled Dyn AI_s (about 6000 cpm), and 100/A of Dyn Am_8 antiserum in assay buffer. After 16-24 h, 1 ml of dextran-coated charcoal in phosphate-buffered saline (2.5 g of charcoal and 0.25 g of dextran per liter) was added to each tube and the tubes were incubated at 4 °C for 10 min, and centrifuged at 2000 g for 20 min. The radioactivity of the supernatant was measured in a Micromedic 4/200 gamma-counter. The RIA sensitivity (20% displacement of trace was less than 8 pg/tube). The antiserum was a gift from E. Weber (Stanford University, Palo Alto, CA). The specificity of the Dyn Al_s RIA has been described 25. The antiserum is directed against the COOH-terminal portion of Dyn AI_8 and does not tolerate COOH-terminal extensions. Dyn At_ 8 antiserum does not recognize Dyn A, Dyn A1_13, Leu-enkephalin, a-neo-endorphin or fl-neo-endorphin; the cross-reactivity with Dyn AI_9 is only 1%.
lodination procedure Synthetic Dyn AI_ 8 (Peninsula Laboratories, San Carlos, CA) was iodinated with Na125I using chloramine-T6. The reaction was stopped with sodium metabisulfite. The radiolabeled peptide purified by chromatography on octadecylsilylsilica cartridges (ODS Silica, Sep Pak C18, Waters Associates), using an increasing gradient of methanol vs 0.01 M HCI/0.1 M acetic acid. RESULTS Ir-Dyn AI_s was detected in all brain areas investigated. The peptide is unevenly distributed. A 10,9fold difference in Dyn AI_ s concentration was measured between the richest (substantia nigra, 673.8 fmol/mg protein) and the poorest (posterior thatamic nucleus, 62.0 fmol/mg protein) brain regions. The neurointermediate lobe is extremely rich in the peptide (4063 fmol/mg protein).
63 TABLE I
Concentration of ir-dynorphin A I_s in telencephalic nuclei of the rat
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Regions
ir-Dynorphin A la concentrations in fmol/mg protein (mean ± S.E.M.)
Molar ratio Dyn At_s:Dyn A
frontal cortex cingulate cortex hippocampus olfactory tubercle nucleus of the diagonal band nucleus aceumbens bed nucleus of the stria terminalis globus pallidus caudate-putamen lateral septal nucleus medial septal nucleus dorsal septal nucleus cortical amygdaloid nucleus basal amygdaloid nucleus medial amygdaloid nucleus lateral amygdaloid nucleus
101.1 ± 23.1 126.2 __+9.9 101.2 + 23.7 141.0 ± 7.9 147.3 + 22.0 195.5 ± 32.6 146.7 ± 26.3 154.9 ± 37.5 106.2 ± 16.3 131.0 ± 22.8 152.2 ± 39.9 132.6 + 32.7 115.3 ± 16.0 205.2 ± 71.5 154.9 ± 29.9 157.3 + 20.1
2.4 2.3 1.6 1.5 1.6 1.9 1.4 2.1 2.0 1.8 1.9 3.0 1.5 2.1 3.4 1.7
Telencephalon (Table I) The telencephalic structures contain low to m o d e r ate concentrations of D y n AI-a. T h e frontal cortex and h i p p o c a m p u s contain relatively low levels of the p e p t i d e (less than 120 fmol/mg protein). Limbic system structures such as a m y g d a l o i d and septal nuclei have m o d e r a t e levels of the p e p t i d e (between 120 and 240 fmol/mg protein). T h e basal ganglia (rostral c o m p o n e n t s of e x t r a p y r a m i d a l system) contain m o d erate (nucleus accumbens, globus pallidus) to low ( c a u d a t e - p u t a m e n ) levels of the peptide.
(5) (4) (6) (4) (6) (5) (5) (5) (6) (6) (5) (5) (6) (5) (6) (6)
Diencephalon (not including the hypothalamus) (Table II) Thalamic structures contain low to m o d e r a t e levels of the peptide. T h e p o s t e r i o r thalamic nucleus, lateral thalamic nucleus, and ventral thalamic nucleus are p o o r while anterior ventral thalamic nucleus, and periventricular nucleus have m o d e r a t e levels as do epithalamic (habenula), subthalamic (zona incerta) and some m e t a t h a l a m i c (medial geniculate b o d y ) structures.
TABLE II
ir-Dynorphin A14 concentrations in the diencephalic (except hypothalamic) nuclei of the rat
17 18 19 20 21 22 23 24 25
Regions
ir-Dynorphin A I_s concentrations in fmol/mg protein (mean ± S,E.M.)
Molar ratio Dyn A l_s:Dyn A
anterior ventral thalamic nucleus periventricular thalamic nucleus ventral thalamic nucleus lateral thalamic nucleus posterior thalamie nucleus lateral geniculate body medial genieulate body habenular nuclei (med. and lat.) zona incerta
160.7 ± 1~.4 139.2 ±20.2 115.6± 6.2 118.6±20.8 62.0± 10.4 80.9 ± 14.9 131.5 ± 19.5 130.2 ± 19.7 166.0 ± 14.2
1.9 1.1 1.7 2.0 1.1 1.1 2.1 1.2 2.2
(4) (6) (4) (4) (4) (5) (5) (4) (4)
64 TABLE III ir-Dynorphin A I~ concentration in nuclei of the hypothalamus Regions
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
medial preoptic nucleus lateral preoptic area medial forebrain bundle (preoptic) periventricular nucleus supraoptic nucleus paraventricular nucleus suprachiasmatic nucleus anterior hypothalamic nucleus median eminence arcuate nucleus ventromedial nucleus dorsomedial nucleus perifornical nucleus posterior hypothalamic nucleus dorsal premamillary nucleus ventral premamillary nucleus mamillary body
Hypothalamus (Table III) T h e h y p o t h a l a m u s is the richest b r a i n r e g i o n in irD y n AI_ 8. V e r y high levels ( a b o v e 240 f m o l / m g protein) w e r e d e t e c t e d in the lateral p r e o p t i c area, v e n -
ir-Dynorphin A I_8 concentrations in fmol/mg protein (mean + S.E.M.) 193.5 + 32.1 565.1 + 64.6 213.3 _+28.6 180.2 +_ 29.1 170.4 _+ 24.5 178.3 __ 31.7 158.4 + 22.1 279.0 __ 41.5 197.6 + 24.7 245.3 _+49.9 197.9 _+45.5 260.0 __ 17.3 168.9 _+ 37.8 ,120.8 _+31.2 179.3 + 24.8 337.9 _+47.2 117.4 _+ 17.3
(6) (6) (4) (6) (5) (6) (5) (5) (5) (4) (5) (4) (6) (5) (6) (5) (6)
Molar ratio Dyn Atm:Dyn A
1.2 3.3 1.5 1.5 2.2 1.4 1.0 1.3 1.1 1.8 1.3 1.3 1.0 1.0 0.9 1.5 0.9
tral p r e m a m i U a r y , a n t e r i o r h y p o t h a l a m i c , d o r s o m e dial a n d a r c u a t e nuclei. T h e rest of the h y p o t h a l a m i c nuclei have m o d e r a t e levels o f t h e p e p t i d e ( b e t w e e n 120 a n d 240 f m o l / m g p r o t e i n ) . T h e m a m i l l a r y b o d y has a relatively low level.
TABLE IV ir-Dynorphin Aim concentration in the mesencephalon and pons Regions
Mesencephalon substantia nigra 43 44 ventral tegmental area 45 interpeduncular nucleus 46 red nucleus superior coUiculus 47 inferior coUiculus 48 periaqueductal grey matter (SGC) 49 dorsal raphe nucleus 50 51 cuneiform nucleus (ret. form.)
ir-Dynorphin A I_s concentrations in fmol/mg protein (mean + S.E.M.)
Molar ratio Dyn A l_e:Dyn A
673.8 + 164.2 (6) 177.2 + 15.8 (5) 183.0 + 30.1 (6) 144.8 + 29.5 (5) 118.9 + 18.0 (4) 84.4 + 15.4 (5) 216.7 + 74.9 (6) 128.2 ___17.0 (5) 130.4 + 20.8 (5)
6.9 2.2 1.9 1.6 3.7 1.3 2.4 1.2 2.1
181.6 + 31.4 133.1 + 29.9 136.5 + 19.4 111.4 + 21.3 113.0 _+ 13.9 116.0 + 24.4 170.7 + 18.3 164.4 + 10.7
1.8 1.1 1.7 2.3 1.6 1.0 2.7 2.2
Pons
52 53 54 55 56 57 58 59
nucleus locus coeruleus parabrachial nuclei (dors. and vent.) dorsal tegmental nuclei pontine reticular nuclei (oral and caud.) pontine nuclei superior olive motor trigeminal nucleus sensory trigeminal nucleus
(5) (6) (5) (3) (5) (6) (4) (4)
65 hemispheres) and nuclei (all 3 nuclei were microdissected together).
Mesencephalon (Table IV) The substantia nigra has the highest concentration of ir-Dyn AI_8 in the brain (673.8 fmol/mg protein). Cell groups surrounding the substantia nigra (red nucleus, ventral tegmental area and interpeduncular nucleus) have much lower levels than the substantia nigra itself. The superior and inferior colliculi have low concentrations of the peptide.
Circumventricular organs Moderate concentrations of ir-Dyn AI_8 are present in the circumventricular organs (Table VI). Pituitary gland The neurointermediate lobe of the pituitary gland contains substantial amounts of ir-Dyn Ax_8 (Table VII).
Pons (Table IV) In general, the tegmentum (parabrachial nuclei, locus coeruleus, and tegmental nuclei) is richer in Dyn AI_8 than the basal pons (pontine nuclei, pontine reticular nuclei and superior olive).
DISCUSSION Using a sensitive and specific radioimmunoassay we have studied the distribution of the opioid peptide, Dyn A1-8, in discrete brain areas and in the neurointermediate lobe of the pituitary gland. Our data show a widespread but uneven distribution of the peptide at all levels of the central nervous system. The molar ratio of Dyn AI_8 to Dyn A in different brain areas is variable. Our results are in good agreement with reports describing the distribution of ir-Dyn At_ s in gross anatomical brain regions 25. There is little immunohisto-
Medulla oblongata (Table V) Most of medullary nuclei have moderate levels of Dyn AI_8. High levels of the peptide were measured in medullary reticular nuclei. Relatively low concentrations of the peptide were found in cochlear nuclei. Cerebellum Ir-Dyn AI_8 is present in the cerebellum but in low concentrations (Table V). There is no difference between the cortex (samples from both vermis and
TABLE V ir-Dynorphin A I_s concentration in medulla oblongata and cerebellum Regions
ir-Dynorphin A I_s concentrations in fraol/mg protein (mean + S.E.M.)
Molar ratio Dyn A l_s:Dyn A
Medulla oblongata 60 nucl. tract, spin. Vth (pars gelatinosa) 61 gracilis nucleus 62 cuneate nucleus 63 medullary reticular nuclei (vent. and dors.) 64 nucleus of the solitary tract (NTS) (medial) 65 motor hypoglossal nucleus 66 nucleus ambiguus 67 lateral reticular nucleus 68 cochlear nuclei (dors. and vent.) 69 lateral vestibular nucleus 70 inferior olive 71 motor facial nucleus 72 gigantocellular reticular nucleus
127.7 + 36.2 (4) 137.7 + 27.5 (4) 163.5 + 52.2 (4) 241.8 + 75.9 (5) 159.7 + 14.5 (5) 197.6 + 86.1 (5) 142.6 + 27.0 (5) 129.8 + 8.7 (4) 117.2 + 25.0 (5) 163.4 + 45.2 (4) 150.2 ± 26.1 (4) 155.6 ± 30.8 (4) 138.7 ± 47.2 (5)
1.6 2.0 2.2 2.4 1.7 1.7 1.5 1.3 1.3 2.1 2.4 2.1 1.8
CerebeUum 73 74
124.1 ± 38.3 (5) 116.1 ± 32.0 (5)
2.3 2.0
cortex nuclei
66 TABLE VI
Concentration of ir-dynorphin A 1-ein the circumventricular organs
75 76 77 78
Regions
ir-Dynorphin A I~ concentrations in fmol/mg protein (mean + S.E.M.)
Molar ratio Dyn Al~:Dyn A
organum vasculosumlaminae terminalis subfornicai organ subcommissural organ area postrema
209.8 _+52.9 (4) 156.7 _ 24.5 (5) 201.2 _+43.3 (5) 156.3 _ 41.0 (4)
1.6 1.7 1.4 0.9
TABLE VII
Content of ir-dynorphin A l_s in neurointermediate lobe of the pituitary gland
Data are mean + S.E.M. Region
ir-Dynorphin A l~ concentrations in fmol/mg protein
Molar ratio Dyn A l_s:Dyn A
neurointermediate lobe
4063.0 + 331.3 (18)
2.0
chemical data on the distribution of ir-Dyn AI_s in the rat central nervous system18, 24. It seems that areas with high concentrations of ir-Dyn Al_8 contain large numbers of ir-dynorphin/neo-endorphin fibers and terminals10,21,24,26. Thus the most dense Dyn A-positive fiber networks were found in the posterior lobe of the pituitary gland, in the substantia nigra and lateral preoptic area which contain substantial amounts of Dyn AI_8. On the other hand, bed nucleus of stria terminalis, caudate-putamen, ventromedial nucleus, supraoptic nucleus, paraventricular nucleus, periaqueductal central grey, parabrachial nuclei, nucleus of the solitary tract, lateral reticular nucleus, areas which all appear to have Dyn A-containing cell bodies, only have moderate levels of ir-Dyn AI_8. This is consistent with the observation that precursors once synthesized are rapidly transported away from cell bodies 1. Thus the precursors and their products do not accumulate in areas with abundant perikarya, but in regions that are densely innervated. There are exceptions to the rule that cell body-rich areas are not remarkably rich in Dyn At_8: ventral premamillary nuclei, anterior hypothalamic nucleus, dorsomedial nucleus, arcuate nucleus, which have among the highest ir-Dyn AI_8 concentrations in the brain contain ir-Dyn A cell bodies10,20,22,23,25. However, all the
above-mentioned nuclei also contain Dyn A-positive nerve fibers or terminals10,21,23,26. In pro-dynorphin the pairs of basic amino acids do not seem to be the only signal for processing by the trypsin-like endopeptidase. For example, Dyn A1_8 must be liberated from its precursor by action of an endopeptidase at a single arginine residue. This should be followed by trimming of the arginine by a carboxypeptidase B-like enzyme. Similarly, Dyn B seems to be generated by cleavage at a single arginine residue 11 as is neurophysin 12and one of the Metenkephalins in the pro-enkephalin precursorZT. About 90% of the brain areas examined have higher concentrations of Dyn AI_8 than Dyn A. There are several areas, where the molar excess of Dyn A1_8 over Dyn A is very large: substantia nigra (6.9 times), superior colliculus (3.7 times), medial amygdaloid nucleus (3.4 times), lateral preoptic area (3.3 times), and dorsal septal nucleus (3 times). About a third of the nuclei examined contained twice as much or more Dyn AI_8 than Dyn A. Among the structures that fall into this class are the frontal and cingulate cortex, globus pallidus, caudate-putamen, supraoptic nucleus, motor trigeminal nucleus and others. There are also several areas where the molar ratio between the two peptides is about one; e.g. supra-
67 chiasmatic nucleus, perifornical nucleus, posterior hypothalamic nucleus and superior olive. Only a few nuclei contain more Dyn A than Dyn A~_8, e.g. dorsal premamillary nucleus, mamillary body and area postrema. The fact that Dyn AI_8 is predominant over Dyn A in most brain areas examined suggests that Dyn A serves chiefly as a precursor. The sum of concentrations of Dyn A and Dyn ml_8 in many brain areas are less than the sum of the concentrations of a and fl-neo-endorphin29,30. This suggests that Dyn A may give rise to a product or products other than Dyn AI_8. One possibility is that Dyn A serves as a precursor for the Leu-enkephalin (just as pro-enkephalin does). This seems to be the case in the substantia nigra for instance (unpublished). The processing of pro-dynorphin into a variety of products, such as Dyn A, Dyn AI_8 and Led-enkephalin yields opioid molecules with different pharmacological properties. Differential processing in brain might
therefore be a mechanism for regulating the action of dynorphinergic neurons. The presence of a large number of ir-Dyn A-positive fibers and terminals and substantial amounts of Dyn Ax..8 in substantia nigra, is particularly suggestive of a role for Dyn AI_8 in motor function. The substantia nigra can serve as a major site for interaction
REFERENCES
10 Khachaturian, H., Watson, S. J., Lewis, M. E., Coy, D., Goldstein, A. and Akil, H., Dynorphin immunohistochemistry in the rat central nervous system, Peptides, 3 (1982) 941-954. 11 Kilpatrick, D. L., Wahlstrom, A., Lahm, H. W., Blacher, R. and Udenfriend, S., Rimorphin, a unique, naturally occurring (Leu)-enkephalin-containingpeptide found in association with dynorphin and a-neo-endorphin, Proc. nat. Acad. Sci. U.S.A., 79 (1982)6480--6483. 12 Land, H., Schutz, G., Schmale, H. and Richter, D., Nudeotide sequence of cloned eDNA encoding bovine arginine vasopressin-neurophysin II precursor, Nature (Lond.), 295 (1982) 299-303. 13 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with Folin phenol reagent, J. biol. Chem., 193 (1951)265--275. 14 Minamino, N., Kangawa, K., Fukuda, A. and Matsuo, H., A new opioid octapeptide related to dynorphin from porcine hypothalamus, Biochem. biophys. Res~ Commun., 95 (1980) 1475-1481. 15 Minamino, N., Kangawa, K., Chino, N., Sakakibara, S. and Matsuo, H., fl-Neo-endorphin, a new hypothalamic 'big' Leu-enkephalin of porcine origin: its purification and the complete amino acid sequence, Biochem. biophys. Res. Commun., 99 (1981) 864-870. 16 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 17 Palkovits, M., Brownstein, M. J. and Zamir, N., Immunoreactive dynorphin and a-neo-endorphin in rat hypothalamo-neurohypophyseal system, Brain Research, 278 (1983) 258--261. 18 Roth, K. A., Weber, E., Barchas, J. D., Chang, D. and Chang, J.-K., Immunoreactive dynorphin-(1-8) and corticotropin-releasing factor in subpopulation of hypothalamic neurons, Science, 219 (1983) 189-191. 19 Seizinger, B. R., H611t,V. and Herz, A., Evidence for the
1 Brownstein, M. J., Russell, J. T. and Gainer, H., Synthesis, transport, and release of posterior pituitary hormones, Science, 207 (1980) 373--380. 2 Chavkin, C. and Goldstein, A., Specific receptor for the opioid peptide dynorphin: structure-activity relationships, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 6543--6547. 3 Corbett, A. D., Paterson, S. J., McKnight, A. T., Magnan, J. and Kosterlitz, H. W., Dynorphinl_a and dynorphint..9 are ligands for the kappa subtype of opiate receptor, Nature (Lond.), 299 (1982) 79-81. 4 Goldstein, A., Tachibana, S., Lowney, L. I., Hunkapiller, M. and Hood, L., Dynorphin-(1-13), an extraordinarily potent opioid peptide, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 6666--6670. 5 Goldstein, A., Fischli, W., Lowney, L. I., Hunkapiller, M. and Hood, L., Porcine pituitary dynorphin: complete amino acid sequence of the biologically active heptadecapeptide, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 7219--7223. 6 Hunter, W. M. and Greenwood, F. C., Preparation of iodine-131 labeled human growth hormone of high specific activity, Nature (Lond.), 194 (1962) 495--496. 7 Kakidani, H., Furutani, Y., Takahashi, M., Noda, M., Morimoto, Y., Hirose, T., Asai, M., Inayama, S., Nankanishi, S. and Numa, S., Cloning and sequence analysis of cDNA for porcine fl-neo-endorphin/dynorphinprecursor, Nature (Lond.), 298 (1982) 245-249. 8 Kangawa, K., Matsuo, H. and Igarashi, M., a-Neo-endorphin: a 'big' Leu-enkephalin with potent opiate activity from porcine hypothalami, Biochem. biophys. Res. Commun., 86 (1979) 153-160. 9 Kangawa, K., Minamino, N., Chino, N., Sakakibara, S. and Matsuo, H., The complete amino acid sequence of a-neo-endorphin, Biochem. biophys. Res. Commun., 99 (1981) 871-878.
between ir-Dyn AI_8 and the dopaminergic system. Dyn AI_8 may also have a role in the neuroendocrine system. Dyn A/Dyn AI_8 perikarya are present in the supraoptic and paraventricular nucleilS, 24, which have been observed also to contain vasopressin 22. Some of these cells give rise to Dyn A/Dyn AI_s fibers in the inner layer of the median eminence and the posterior lobe of the pituitary17,21,23,26. In addition the lateral preoptic area, medial preoptic area, suprachiasmatic nucleus, anterior hypothalamic nucleus and arcuate nucleus may represent sites which Dyn AI_s could affect the neuroendocrine system.
68
20
21
22
23
24
25
occurrence of the opioid octapeptide dynorphin-(1-8) in the neurointermediate pituitary of rats, Biochem. biophys. Res. Commun., 102 (1981) 197-205. Udenfriend, S. and Kilpatrick, D. L., Biochemistry of the enkephalins and enkephalin-containing peptides, Arch. Biochem. Biophys., 221 (1983)309-323. Vincent, S. R., HOkfelt, T., Christensson, I. and Terenius, L., Dynorphin-immunoreactive neurons in the central nervous system of the rat, Neurosci. Lett., 33 (1982) 185-190. Watson, S. J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, E., Nilaver, G. and van Wimersma Greidanus, Tj. B., Dynorphin and vasopressin: common localization in magnocellular neurons, Science, 216 (1982) 85-87, Weber, E., Roth, K. A. and Barchas, J. D., Immunohistochemical distribution of ct-neo-endorphin/dynorphin neuronal systems in rat brain: evidence for colocalization, Proc, nat. Acad. Sci. U.S.A., 79 (1982) 3062-3066. Weber, E., Roth, K. A., Evans, C..1., Chang, K.-J. and Barchas, J. D., Immunohistochemical localization of dynorphin-(1-8) in hypothalamic magnocellular neurons: evidence for absence for proenkephalin, Life Sci., 31 (1982) 1761-1764. Weber, E., Evans, C..1. and Barchas, J. D., Predominance
26
27
28
29
30
of the amino-terminal octapeptide fragment of dynorphin in rat brain regions, Nature (Lond.), 299 (1982) 77-79. Weber, E. and Barchas, J. D., Immunohistochemical distribution of dynorphin B in rat brain: relation to dynorphin A and a-neo-endorphin system, Proc. nat. Acad. Sci. U.S.A., 80 (1983) 1125-1129. Weber, E., Evans, C. J., Barchas, J. D., Bohlen, P. and Esch, F., Identification and characterization of metorphamide, a novel amidated opioid octapeptide from bovine brain, International Narcotic Research Conference (INRC) Garmisch-Partenkirchen, F.R.G., June 26--July 1, 1983 (Abstr. L-I). Zamir, N., Palkovits, M. and Brownstein, M. J., Distribution of immunoreactive dynorphin in the central nervous system of the rat, Brain Research, 280 (1983) 81-93. Zamir, N., Palkovits, M. and Brownstein, M. J., Distribution of immunoreactive a-neo-endorphin in the central nervous system of the rat, J. Neurosci., (submitted). Zamir, N., Palkovits, M. and Brownstein, M. J., Distribution of immunoreactive fl-neo-endorphin in discrete nuclei of the rat brain: comparison with a-neo-endorphin, J. Neurosci., in press.
61
BILE 10222
Distribution of Immunoreactive Dynorphin AI_.8 in Discrete Nuclei of the Rat Brain: Comparison with Dynorphin A NADAV ZAMIR, MIKLOS PALKOVITS and MICHAEL J. BROWNSTEIN Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20205 (U.S.A.)
(Accepted January 3rd, 1984) Key words: dynorphin AI_8 - dynorphln A - - Leu-enkephalin - - RIA - - rat brain nuclei - - posterior pituitary
The distribution of immunoreactive (ir)-dynorphin A1..s (Dyn A1..s) in 78 microdissected rat brain areas as well as in the neurointermediate lobe of pituitary gland was determined using a highly specific radioimmunoassay. The highest concentrations of Dyn At..a in brain were found in substantia nigra (673.8 fmol/mg protein) and lateral preoptic area (565.1 fmol/mgprotein). High concentrations of ir-Dyn A1..s (> 240 fmol/mg protein) were found in 5 nuclei: ventral premamillary nucleus,:anterior hypothalamic nucleus, dorsomedial nucleus, areuate nucleus, and medullary reticular nuclei. Moderate concentrations of the peptide (between 120 and 240 fmol/mg protein) were found in 55 brain nuclei such as septal and amygdaloid nuclei, most diencephalic structures, mesencephalic nuclei, loons and medulla oblongata nuclei and others. Low concentrations of ir-Dyn A1..s (< 120 frnoFmgprotein) were found in 16 regions, e.g. frontal cortex, hlppocampus, caudate-putamen cortical amygdaloid nucleus, several thaiamic nuclei, mamillary body superior and inferior colliculi, cerebellar nuclei and others. The posterior thalamic nucleus has the lowest ir-Dyn AI_s concentration (62.0 fmol/mg protein). The neurointermediate lobe of the pituitary gland is extremely rich in ir-Dyn AI..a (4063.0 fmol/mg protein).
INTRODUCTION Dynorphin A1-8 (Dyn A1-8) is an endogenous opioid peptide which is an amino terminal fragment of dynorphin (Dyn A)4, 5,14,19. The structural relationship of the two peptides is illustrated below:
(a) dynorphin A: Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro1 5 8 -Lys-Leu-Lys-Trp-Asp--Asn-Gln 17 (b) dynorphin Al-s: Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile 1 5 8 It contains the amino acid sequence of Leu-enkeph-
alin at its amino terminus, and is a potent opiate agonist in the in vitro guinea pig myenteric plexus longitudinal muscle bioassay. It appears to be a highly selective ligand for the kappa opiate binding site2, 3. Dyn A and Dyn AI_8 are parts of the same precursor that gives rise to the neo-endorphinsS,9,15 and dynorphin B n. The amino acid sequence of this precursor has been deduced from the structure of D N A complementary to pro-dynorphin m R N A from porcine hypothalamus 7. Prodynorphin contains three Leuenkephalin sequences that form the N-termini of a-neo-endorphin, Dyn A, and Dyn B, respectively7. a-Neo-endorphin and the two dynorphins are flanked on either side by basic residues which serve as processing signals. These basic residues seem to be cleaved in preference to bases within a-neo-endorphin and the dynorphins. The latter can be cleaved, however. Presumably the A r g - P r o bond in Dyn A is severed and the Arg removed by carboxypeptidase B
Correspondence: N. Zamir, Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20205, U.S.A.
62 to yield Dyn AI_ s. Furthermore, it seems likely that Leu-enkephalin can also be liberated from Dyn A. Immunohistochemical studies have shown that perikarya, nerve fibers and terminals containing Dyn A are widely distributed throughout the central nervous system10.21,23. The distribution of Dyn AI_S, measured by RIA, in gross brain areas has been reported 25. The hypothalamus, striatum and midbrain are regions rich in the peptide; the hippocampus, medulla oblongata, and cortex contain moderate amounts of the peptide. The cerebellum has very little Dyn AI_8 (ref. 25). In this paper we describe the topographical distribution of ir-Dyn AI_s among 78 microdissected brain areas as well as neurointermediate lobe of the pituitary gland. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (Zivic-MiUer Laboratories, Allison Park, PA), weighing 220-250 g were housed under alternate 12-h periods of dark and light (lights on from 06.00 to 18.00 h) and were given standard rat chow and tap water ad libitum.
Tissue preparation and extraction The animals were killed by decapitation between 08.00 and 10.00 h. The brains were quickly removed and frozen on dry-ice. Brain areas were removed from 300/zm thick frozen coronal sections cut in a cryostat at --10 °C t6. Tissue from each microdissected area was pooled from two animals. Dyn AI_s and Dyn A were assayed in the same tissue samples. The distribution of the latter has been reported earlier 28. The pituitary gland was immediately removed and dissected into neurointermediate and anterior lobes. Tissue samples were placed in 1.5 ml conical Eppendorf tubes containing 200/xl of 0.1 N HC1 and transferred to a boiling water bath for 10 min. The tissue samples were then homogenized by sonication and 20/A aliquots of the homogenates were removed for protein determination t3. The extracts were centrifuged at 2000 g for 10 min at 4 °C, The supernatants were transferred to 12 x 75 mm polypropylene tubes and evaporated to dryness in a vacuum centrifuge.
Radioimmunoassay and specificity of the antiserum Samples were rehydrated in phosphate-buffered saline (pH 7.6) containing 0.1% gelatin, 0.1% bovine serum albumin, 0.1% Triton X-100 and 0.01% merthiolate. The antiserum was used at a final dilution of 1:120,000, which resulted in binding of 30-45% trace. Each sample was incubated at 4 °C in a 500/A volume that contained 300/A of sample in assay buffer, 100 ktl of [125I]labeled Dyn AI_s (about 6000 cpm), and 100/A of Dyn Am_8 antiserum in assay buffer. After 16-24 h, 1 ml of dextran-coated charcoal in phosphate-buffered saline (2.5 g of charcoal and 0.25 g of dextran per liter) was added to each tube and the tubes were incubated at 4 °C for 10 min, and centrifuged at 2000 g for 20 min. The radioactivity of the supernatant was measured in a Micromedic 4/200 gamma-counter. The RIA sensitivity (20% displacement of trace was less than 8 pg/tube). The antiserum was a gift from E. Weber (Stanford University, Palo Alto, CA). The specificity of the Dyn Al_s RIA has been described 25. The antiserum is directed against the COOH-terminal portion of Dyn AI_8 and does not tolerate COOH-terminal extensions. Dyn At_ 8 antiserum does not recognize Dyn A, Dyn A1_13, Leu-enkephalin, a-neo-endorphin or fl-neo-endorphin; the cross-reactivity with Dyn AI_9 is only 1%.
lodination procedure Synthetic Dyn AI_ 8 (Peninsula Laboratories, San Carlos, CA) was iodinated with Na125I using chloramine-T6. The reaction was stopped with sodium metabisulfite. The radiolabeled peptide purified by chromatography on octadecylsilylsilica cartridges (ODS Silica, Sep Pak C18, Waters Associates), using an increasing gradient of methanol vs 0.01 M HCI/0.1 M acetic acid. RESULTS Ir-Dyn AI_s was detected in all brain areas investigated. The peptide is unevenly distributed. A 10,9fold difference in Dyn AI_ s concentration was measured between the richest (substantia nigra, 673.8 fmol/mg protein) and the poorest (posterior thatamic nucleus, 62.0 fmol/mg protein) brain regions. The neurointermediate lobe is extremely rich in the peptide (4063 fmol/mg protein).
63 TABLE I
Concentration of ir-dynorphin A I_s in telencephalic nuclei of the rat
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Regions
ir-Dynorphin A la concentrations in fmol/mg protein (mean ± S.E.M.)
Molar ratio Dyn At_s:Dyn A
frontal cortex cingulate cortex hippocampus olfactory tubercle nucleus of the diagonal band nucleus aceumbens bed nucleus of the stria terminalis globus pallidus caudate-putamen lateral septal nucleus medial septal nucleus dorsal septal nucleus cortical amygdaloid nucleus basal amygdaloid nucleus medial amygdaloid nucleus lateral amygdaloid nucleus
101.1 ± 23.1 126.2 __+9.9 101.2 + 23.7 141.0 ± 7.9 147.3 + 22.0 195.5 ± 32.6 146.7 ± 26.3 154.9 ± 37.5 106.2 ± 16.3 131.0 ± 22.8 152.2 ± 39.9 132.6 + 32.7 115.3 ± 16.0 205.2 ± 71.5 154.9 ± 29.9 157.3 + 20.1
2.4 2.3 1.6 1.5 1.6 1.9 1.4 2.1 2.0 1.8 1.9 3.0 1.5 2.1 3.4 1.7
Telencephalon (Table I) The telencephalic structures contain low to m o d e r ate concentrations of D y n AI-a. T h e frontal cortex and h i p p o c a m p u s contain relatively low levels of the p e p t i d e (less than 120 fmol/mg protein). Limbic system structures such as a m y g d a l o i d and septal nuclei have m o d e r a t e levels of the p e p t i d e (between 120 and 240 fmol/mg protein). T h e basal ganglia (rostral c o m p o n e n t s of e x t r a p y r a m i d a l system) contain m o d erate (nucleus accumbens, globus pallidus) to low ( c a u d a t e - p u t a m e n ) levels of the peptide.
(5) (4) (6) (4) (6) (5) (5) (5) (6) (6) (5) (5) (6) (5) (6) (6)
Diencephalon (not including the hypothalamus) (Table II) Thalamic structures contain low to m o d e r a t e levels of the peptide. T h e p o s t e r i o r thalamic nucleus, lateral thalamic nucleus, and ventral thalamic nucleus are p o o r while anterior ventral thalamic nucleus, and periventricular nucleus have m o d e r a t e levels as do epithalamic (habenula), subthalamic (zona incerta) and some m e t a t h a l a m i c (medial geniculate b o d y ) structures.
TABLE II
ir-Dynorphin A14 concentrations in the diencephalic (except hypothalamic) nuclei of the rat
17 18 19 20 21 22 23 24 25
Regions
ir-Dynorphin A I_s concentrations in fmol/mg protein (mean ± S,E.M.)
Molar ratio Dyn A l_s:Dyn A
anterior ventral thalamic nucleus periventricular thalamic nucleus ventral thalamic nucleus lateral thalamic nucleus posterior thalamie nucleus lateral geniculate body medial genieulate body habenular nuclei (med. and lat.) zona incerta
160.7 ± 1~.4 139.2 ±20.2 115.6± 6.2 118.6±20.8 62.0± 10.4 80.9 ± 14.9 131.5 ± 19.5 130.2 ± 19.7 166.0 ± 14.2
1.9 1.1 1.7 2.0 1.1 1.1 2.1 1.2 2.2
(4) (6) (4) (4) (4) (5) (5) (4) (4)
64 TABLE III ir-Dynorphin A I~ concentration in nuclei of the hypothalamus Regions
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
medial preoptic nucleus lateral preoptic area medial forebrain bundle (preoptic) periventricular nucleus supraoptic nucleus paraventricular nucleus suprachiasmatic nucleus anterior hypothalamic nucleus median eminence arcuate nucleus ventromedial nucleus dorsomedial nucleus perifornical nucleus posterior hypothalamic nucleus dorsal premamillary nucleus ventral premamillary nucleus mamillary body
Hypothalamus (Table III) T h e h y p o t h a l a m u s is the richest b r a i n r e g i o n in irD y n AI_ 8. V e r y high levels ( a b o v e 240 f m o l / m g protein) w e r e d e t e c t e d in the lateral p r e o p t i c area, v e n -
ir-Dynorphin A I_8 concentrations in fmol/mg protein (mean + S.E.M.) 193.5 + 32.1 565.1 + 64.6 213.3 _+28.6 180.2 +_ 29.1 170.4 _+ 24.5 178.3 __ 31.7 158.4 + 22.1 279.0 __ 41.5 197.6 + 24.7 245.3 _+49.9 197.9 _+45.5 260.0 __ 17.3 168.9 _+ 37.8 ,120.8 _+31.2 179.3 + 24.8 337.9 _+47.2 117.4 _+ 17.3
(6) (6) (4) (6) (5) (6) (5) (5) (5) (4) (5) (4) (6) (5) (6) (5) (6)
Molar ratio Dyn Atm:Dyn A
1.2 3.3 1.5 1.5 2.2 1.4 1.0 1.3 1.1 1.8 1.3 1.3 1.0 1.0 0.9 1.5 0.9
tral p r e m a m i U a r y , a n t e r i o r h y p o t h a l a m i c , d o r s o m e dial a n d a r c u a t e nuclei. T h e rest of the h y p o t h a l a m i c nuclei have m o d e r a t e levels o f t h e p e p t i d e ( b e t w e e n 120 a n d 240 f m o l / m g p r o t e i n ) . T h e m a m i l l a r y b o d y has a relatively low level.
TABLE IV ir-Dynorphin Aim concentration in the mesencephalon and pons Regions
Mesencephalon substantia nigra 43 44 ventral tegmental area 45 interpeduncular nucleus 46 red nucleus superior coUiculus 47 inferior coUiculus 48 periaqueductal grey matter (SGC) 49 dorsal raphe nucleus 50 51 cuneiform nucleus (ret. form.)
ir-Dynorphin A I_s concentrations in fmol/mg protein (mean + S.E.M.)
Molar ratio Dyn A l_e:Dyn A
673.8 + 164.2 (6) 177.2 + 15.8 (5) 183.0 + 30.1 (6) 144.8 + 29.5 (5) 118.9 + 18.0 (4) 84.4 + 15.4 (5) 216.7 + 74.9 (6) 128.2 ___17.0 (5) 130.4 + 20.8 (5)
6.9 2.2 1.9 1.6 3.7 1.3 2.4 1.2 2.1
181.6 + 31.4 133.1 + 29.9 136.5 + 19.4 111.4 + 21.3 113.0 _+ 13.9 116.0 + 24.4 170.7 + 18.3 164.4 + 10.7
1.8 1.1 1.7 2.3 1.6 1.0 2.7 2.2
Pons
52 53 54 55 56 57 58 59
nucleus locus coeruleus parabrachial nuclei (dors. and vent.) dorsal tegmental nuclei pontine reticular nuclei (oral and caud.) pontine nuclei superior olive motor trigeminal nucleus sensory trigeminal nucleus
(5) (6) (5) (3) (5) (6) (4) (4)
65 hemispheres) and nuclei (all 3 nuclei were microdissected together).
Mesencephalon (Table IV) The substantia nigra has the highest concentration of ir-Dyn AI_8 in the brain (673.8 fmol/mg protein). Cell groups surrounding the substantia nigra (red nucleus, ventral tegmental area and interpeduncular nucleus) have much lower levels than the substantia nigra itself. The superior and inferior colliculi have low concentrations of the peptide.
Circumventricular organs Moderate concentrations of ir-Dyn AI_8 are present in the circumventricular organs (Table VI). Pituitary gland The neurointermediate lobe of the pituitary gland contains substantial amounts of ir-Dyn Ax_8 (Table VII).
Pons (Table IV) In general, the tegmentum (parabrachial nuclei, locus coeruleus, and tegmental nuclei) is richer in Dyn AI_8 than the basal pons (pontine nuclei, pontine reticular nuclei and superior olive).
DISCUSSION Using a sensitive and specific radioimmunoassay we have studied the distribution of the opioid peptide, Dyn A1-8, in discrete brain areas and in the neurointermediate lobe of the pituitary gland. Our data show a widespread but uneven distribution of the peptide at all levels of the central nervous system. The molar ratio of Dyn AI_8 to Dyn A in different brain areas is variable. Our results are in good agreement with reports describing the distribution of ir-Dyn At_ s in gross anatomical brain regions 25. There is little immunohisto-
Medulla oblongata (Table V) Most of medullary nuclei have moderate levels of Dyn AI_8. High levels of the peptide were measured in medullary reticular nuclei. Relatively low concentrations of the peptide were found in cochlear nuclei. Cerebellum Ir-Dyn AI_8 is present in the cerebellum but in low concentrations (Table V). There is no difference between the cortex (samples from both vermis and
TABLE V ir-Dynorphin A I_s concentration in medulla oblongata and cerebellum Regions
ir-Dynorphin A I_s concentrations in fraol/mg protein (mean + S.E.M.)
Molar ratio Dyn A l_s:Dyn A
Medulla oblongata 60 nucl. tract, spin. Vth (pars gelatinosa) 61 gracilis nucleus 62 cuneate nucleus 63 medullary reticular nuclei (vent. and dors.) 64 nucleus of the solitary tract (NTS) (medial) 65 motor hypoglossal nucleus 66 nucleus ambiguus 67 lateral reticular nucleus 68 cochlear nuclei (dors. and vent.) 69 lateral vestibular nucleus 70 inferior olive 71 motor facial nucleus 72 gigantocellular reticular nucleus
127.7 + 36.2 (4) 137.7 + 27.5 (4) 163.5 + 52.2 (4) 241.8 + 75.9 (5) 159.7 + 14.5 (5) 197.6 + 86.1 (5) 142.6 + 27.0 (5) 129.8 + 8.7 (4) 117.2 + 25.0 (5) 163.4 + 45.2 (4) 150.2 ± 26.1 (4) 155.6 ± 30.8 (4) 138.7 ± 47.2 (5)
1.6 2.0 2.2 2.4 1.7 1.7 1.5 1.3 1.3 2.1 2.4 2.1 1.8
CerebeUum 73 74
124.1 ± 38.3 (5) 116.1 ± 32.0 (5)
2.3 2.0
cortex nuclei
66 TABLE VI
Concentration of ir-dynorphin A 1-ein the circumventricular organs
75 76 77 78
Regions
ir-Dynorphin A I~ concentrations in fmol/mg protein (mean + S.E.M.)
Molar ratio Dyn Al~:Dyn A
organum vasculosumlaminae terminalis subfornicai organ subcommissural organ area postrema
209.8 _+52.9 (4) 156.7 _ 24.5 (5) 201.2 _+43.3 (5) 156.3 _ 41.0 (4)
1.6 1.7 1.4 0.9
TABLE VII
Content of ir-dynorphin A l_s in neurointermediate lobe of the pituitary gland
Data are mean + S.E.M. Region
ir-Dynorphin A l~ concentrations in fmol/mg protein
Molar ratio Dyn A l_s:Dyn A
neurointermediate lobe
4063.0 + 331.3 (18)
2.0
chemical data on the distribution of ir-Dyn AI_s in the rat central nervous system18, 24. It seems that areas with high concentrations of ir-Dyn Al_8 contain large numbers of ir-dynorphin/neo-endorphin fibers and terminals10,21,24,26. Thus the most dense Dyn A-positive fiber networks were found in the posterior lobe of the pituitary gland, in the substantia nigra and lateral preoptic area which contain substantial amounts of Dyn AI_8. On the other hand, bed nucleus of stria terminalis, caudate-putamen, ventromedial nucleus, supraoptic nucleus, paraventricular nucleus, periaqueductal central grey, parabrachial nuclei, nucleus of the solitary tract, lateral reticular nucleus, areas which all appear to have Dyn A-containing cell bodies, only have moderate levels of ir-Dyn AI_8. This is consistent with the observation that precursors once synthesized are rapidly transported away from cell bodies 1. Thus the precursors and their products do not accumulate in areas with abundant perikarya, but in regions that are densely innervated. There are exceptions to the rule that cell body-rich areas are not remarkably rich in Dyn At_8: ventral premamillary nuclei, anterior hypothalamic nucleus, dorsomedial nucleus, arcuate nucleus, which have among the highest ir-Dyn AI_8 concentrations in the brain contain ir-Dyn A cell bodies10,20,22,23,25. However, all the
above-mentioned nuclei also contain Dyn A-positive nerve fibers or terminals10,21,23,26. In pro-dynorphin the pairs of basic amino acids do not seem to be the only signal for processing by the trypsin-like endopeptidase. For example, Dyn A1_8 must be liberated from its precursor by action of an endopeptidase at a single arginine residue. This should be followed by trimming of the arginine by a carboxypeptidase B-like enzyme. Similarly, Dyn B seems to be generated by cleavage at a single arginine residue 11 as is neurophysin 12and one of the Metenkephalins in the pro-enkephalin precursorZT. About 90% of the brain areas examined have higher concentrations of Dyn AI_8 than Dyn A. There are several areas, where the molar excess of Dyn A1_8 over Dyn A is very large: substantia nigra (6.9 times), superior colliculus (3.7 times), medial amygdaloid nucleus (3.4 times), lateral preoptic area (3.3 times), and dorsal septal nucleus (3 times). About a third of the nuclei examined contained twice as much or more Dyn AI_8 than Dyn A. Among the structures that fall into this class are the frontal and cingulate cortex, globus pallidus, caudate-putamen, supraoptic nucleus, motor trigeminal nucleus and others. There are also several areas where the molar ratio between the two peptides is about one; e.g. supra-
67 chiasmatic nucleus, perifornical nucleus, posterior hypothalamic nucleus and superior olive. Only a few nuclei contain more Dyn A than Dyn A~_8, e.g. dorsal premamillary nucleus, mamillary body and area postrema. The fact that Dyn AI_8 is predominant over Dyn A in most brain areas examined suggests that Dyn A serves chiefly as a precursor. The sum of concentrations of Dyn A and Dyn ml_8 in many brain areas are less than the sum of the concentrations of a and fl-neo-endorphin29,30. This suggests that Dyn A may give rise to a product or products other than Dyn AI_8. One possibility is that Dyn A serves as a precursor for the Leu-enkephalin (just as pro-enkephalin does). This seems to be the case in the substantia nigra for instance (unpublished). The processing of pro-dynorphin into a variety of products, such as Dyn A, Dyn AI_8 and Led-enkephalin yields opioid molecules with different pharmacological properties. Differential processing in brain might
therefore be a mechanism for regulating the action of dynorphinergic neurons. The presence of a large number of ir-Dyn A-positive fibers and terminals and substantial amounts of Dyn Ax..8 in substantia nigra, is particularly suggestive of a role for Dyn AI_8 in motor function. The substantia nigra can serve as a major site for interaction
REFERENCES
10 Khachaturian, H., Watson, S. J., Lewis, M. E., Coy, D., Goldstein, A. and Akil, H., Dynorphin immunohistochemistry in the rat central nervous system, Peptides, 3 (1982) 941-954. 11 Kilpatrick, D. L., Wahlstrom, A., Lahm, H. W., Blacher, R. and Udenfriend, S., Rimorphin, a unique, naturally occurring (Leu)-enkephalin-containingpeptide found in association with dynorphin and a-neo-endorphin, Proc. nat. Acad. Sci. U.S.A., 79 (1982)6480--6483. 12 Land, H., Schutz, G., Schmale, H. and Richter, D., Nudeotide sequence of cloned eDNA encoding bovine arginine vasopressin-neurophysin II precursor, Nature (Lond.), 295 (1982) 299-303. 13 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with Folin phenol reagent, J. biol. Chem., 193 (1951)265--275. 14 Minamino, N., Kangawa, K., Fukuda, A. and Matsuo, H., A new opioid octapeptide related to dynorphin from porcine hypothalamus, Biochem. biophys. Res~ Commun., 95 (1980) 1475-1481. 15 Minamino, N., Kangawa, K., Chino, N., Sakakibara, S. and Matsuo, H., fl-Neo-endorphin, a new hypothalamic 'big' Leu-enkephalin of porcine origin: its purification and the complete amino acid sequence, Biochem. biophys. Res. Commun., 99 (1981) 864-870. 16 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 17 Palkovits, M., Brownstein, M. J. and Zamir, N., Immunoreactive dynorphin and a-neo-endorphin in rat hypothalamo-neurohypophyseal system, Brain Research, 278 (1983) 258--261. 18 Roth, K. A., Weber, E., Barchas, J. D., Chang, D. and Chang, J.-K., Immunoreactive dynorphin-(1-8) and corticotropin-releasing factor in subpopulation of hypothalamic neurons, Science, 219 (1983) 189-191. 19 Seizinger, B. R., H611t,V. and Herz, A., Evidence for the
1 Brownstein, M. J., Russell, J. T. and Gainer, H., Synthesis, transport, and release of posterior pituitary hormones, Science, 207 (1980) 373--380. 2 Chavkin, C. and Goldstein, A., Specific receptor for the opioid peptide dynorphin: structure-activity relationships, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 6543--6547. 3 Corbett, A. D., Paterson, S. J., McKnight, A. T., Magnan, J. and Kosterlitz, H. W., Dynorphinl_a and dynorphint..9 are ligands for the kappa subtype of opiate receptor, Nature (Lond.), 299 (1982) 79-81. 4 Goldstein, A., Tachibana, S., Lowney, L. I., Hunkapiller, M. and Hood, L., Dynorphin-(1-13), an extraordinarily potent opioid peptide, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 6666--6670. 5 Goldstein, A., Fischli, W., Lowney, L. I., Hunkapiller, M. and Hood, L., Porcine pituitary dynorphin: complete amino acid sequence of the biologically active heptadecapeptide, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 7219--7223. 6 Hunter, W. M. and Greenwood, F. C., Preparation of iodine-131 labeled human growth hormone of high specific activity, Nature (Lond.), 194 (1962) 495--496. 7 Kakidani, H., Furutani, Y., Takahashi, M., Noda, M., Morimoto, Y., Hirose, T., Asai, M., Inayama, S., Nankanishi, S. and Numa, S., Cloning and sequence analysis of cDNA for porcine fl-neo-endorphin/dynorphinprecursor, Nature (Lond.), 298 (1982) 245-249. 8 Kangawa, K., Matsuo, H. and Igarashi, M., a-Neo-endorphin: a 'big' Leu-enkephalin with potent opiate activity from porcine hypothalami, Biochem. biophys. Res. Commun., 86 (1979) 153-160. 9 Kangawa, K., Minamino, N., Chino, N., Sakakibara, S. and Matsuo, H., The complete amino acid sequence of a-neo-endorphin, Biochem. biophys. Res. Commun., 99 (1981) 871-878.
between ir-Dyn AI_8 and the dopaminergic system. Dyn AI_8 may also have a role in the neuroendocrine system. Dyn A/Dyn AI_8 perikarya are present in the supraoptic and paraventricular nucleilS, 24, which have been observed also to contain vasopressin 22. Some of these cells give rise to Dyn A/Dyn AI_s fibers in the inner layer of the median eminence and the posterior lobe of the pituitary17,21,23,26. In addition the lateral preoptic area, medial preoptic area, suprachiasmatic nucleus, anterior hypothalamic nucleus and arcuate nucleus may represent sites which Dyn AI_s could affect the neuroendocrine system.
68
20
21
22
23
24
25
occurrence of the opioid octapeptide dynorphin-(1-8) in the neurointermediate pituitary of rats, Biochem. biophys. Res. Commun., 102 (1981) 197-205. Udenfriend, S. and Kilpatrick, D. L., Biochemistry of the enkephalins and enkephalin-containing peptides, Arch. Biochem. Biophys., 221 (1983)309-323. Vincent, S. R., HOkfelt, T., Christensson, I. and Terenius, L., Dynorphin-immunoreactive neurons in the central nervous system of the rat, Neurosci. Lett., 33 (1982) 185-190. Watson, S. J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, E., Nilaver, G. and van Wimersma Greidanus, Tj. B., Dynorphin and vasopressin: common localization in magnocellular neurons, Science, 216 (1982) 85-87, Weber, E., Roth, K. A. and Barchas, J. D., Immunohistochemical distribution of ct-neo-endorphin/dynorphin neuronal systems in rat brain: evidence for colocalization, Proc, nat. Acad. Sci. U.S.A., 79 (1982) 3062-3066. Weber, E., Roth, K. A., Evans, C..1., Chang, K.-J. and Barchas, J. D., Immunohistochemical localization of dynorphin-(1-8) in hypothalamic magnocellular neurons: evidence for absence for proenkephalin, Life Sci., 31 (1982) 1761-1764. Weber, E., Evans, C..1. and Barchas, J. D., Predominance
26
27
28
29
30
of the amino-terminal octapeptide fragment of dynorphin in rat brain regions, Nature (Lond.), 299 (1982) 77-79. Weber, E. and Barchas, J. D., Immunohistochemical distribution of dynorphin B in rat brain: relation to dynorphin A and a-neo-endorphin system, Proc. nat. Acad. Sci. U.S.A., 80 (1983) 1125-1129. Weber, E., Evans, C. J., Barchas, J. D., Bohlen, P. and Esch, F., Identification and characterization of metorphamide, a novel amidated opioid octapeptide from bovine brain, International Narcotic Research Conference (INRC) Garmisch-Partenkirchen, F.R.G., June 26--July 1, 1983 (Abstr. L-I). Zamir, N., Palkovits, M. and Brownstein, M. J., Distribution of immunoreactive dynorphin in the central nervous system of the rat, Brain Research, 280 (1983) 81-93. Zamir, N., Palkovits, M. and Brownstein, M. J., Distribution of immunoreactive a-neo-endorphin in the central nervous system of the rat, J. Neurosci., (submitted). Zamir, N., Palkovits, M. and Brownstein, M. J., Distribution of immunoreactive fl-neo-endorphin in discrete nuclei of the rat brain: comparison with a-neo-endorphin, J. Neurosci., in press.