Prodynorphin-Derived Peptide Expression in Primate Cortex and Striatum
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Neuropeptides (1994) 21, 211-284 $:I Longman Group Ltd 1994 Prodynorphin-derived Peptide Expression in Primate Cortex and Striatum D. J. HEALY and J. H. MEADOR-WOODRUFF Department of Psychiatry, Mental Health Research institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109-0720, USA (Reprint requests to DJH) Abstract-The distributions of four prodynorphin-derived peptides, dynorphin A (l-171, dynorphin A (I-81, dynorphin B, and cr-neo-endorphin were determined in 10 cortical regions and the striatum of the old world monkey (Macaca nemestrina). a-neo-endorphin was the most abundant peptide in both cortex and striatum. The concentrations of all four peptides were significantly greater in the striatum compared to the cortex. In general, concentrations of each peptide tended to be higher in allocortex than in neocortex. Possible inter- and intradomain processing differences, as estimated by ratios of these peptides, did not vary within cortex, but the intradomain peptide ratio, dyn A (l-17)/dyn A (l-81, was significantly greater in cortex than in striatum. These results indicate that prodynorphin is, in some ways, uniquely processed in the primate. Particularly unusual is the relatively low abundance of prodynorphin-derived products in the cortex, in the face of moderately high levels of kappa opiate receptor expression. Introduction morphin, and neoendorphin domains.’ Dynorphin A, which is dynorphin A (l-1 7) (dyn A (l-l 7)), can Prodynorphin, proenkephalin, and pro- be further processed into dynorphin A (l-8) (dyn A opiomelanocortin are the 3 propeptides that serve (l-8)) (Fig. 1).14.17Dyn A (l-1 7) has greater affinity as precursors to endogenous opioid peptides. These for the K opiate receptor than dyn A (l-8).’ The 3 precursors make a number of peptide ligands leumorphin domain can be further processed into with affinities at opiate receptors. Prodynorphin dynorphin B (dyn B).” Dyn B has an affinity for produces ligands with varying affinities for the p the K opiate receptor similar to dyn A (1-17).9 The and 6 opiate receptors,’ but differs from pro- neoendorphin domain, which is a-neo-endorphin opiomelanocortin and proenkephalin by also pro- (tx-neo) can be further processed into p-neo- ducing peptides with affinity for the K opiate endorphin (j?-neo).6. I3 receptor.’ 3 domains have been identified in the a-neo has an affinity for the K opiate receptor similar to dyn B and dyn A (l- prodynorphin molecule, the dynorphin A, leu- 17).9 These 3 molecules also bind to p and 6 opiate receptors, but these affinities are 6 to 20 times less Date received 22 March 1994 Date revised 11 April 1994 than their affinities for the K opiate receptor.’ The Date accepted 18 April 1994 extent of how each domain is processed may thus 277 278 NEUROPEPTIDES a-neo ) +Dyn A (1-17)b Leumorphin ,Bneo, + “ynB ) Fig. 1 Schematic representation of the prodynorphin molecule. There are 3 domains, the neoendorphin domain (a-neoendorphin (a-neo) potentially processed into /I-neoendorphin (/I-neo)), the dynorphin A (dyn A) domain (dyn A (1-17) potentially processed into dyn A (l-8)), and the Ieumorphin domain potentially processed into dynorphin B (dyn B)). alter the functional opioid selectivity of the parallels the distribution of both prodynorphin domain.2 However, the physiological activity of fibers and IC receptor binding in rodent brain. prodyn is not only determined by expression and Further, concentrations of various prodynorphin processing of these domains, but also by the specific derived peptides appear to be similar in the rodent anatomical locations of where the peptides (and striatum and cortex.” In those regions in which associated opiate receptors) are expressed. fairly detailed examination of the various pro- Prodynorphin has been most extensively studied dynorphin derived peptides have been performed in the rodent. A specific pattern of distribution of (primarily rat striatum and cortex), several con- prodynorphin-containing cells and fibers in rodent sistent observations have been made. In the rat brain has been found using immunocytochemistry, brain, a-neo is, by far, the most abundant peptide with prod$norphin being visualized in the caudate- present. The A and B domains are present in much putamen, substantia nigra, hippocampus, hypo- lower concentrations, and the A domain is pref- thalamus, amygdala, parabrachial nucleus, nucleus erentially processed to dyn A ( l-8). tractus solitarius, substantia grisea centralis, as well The primate prodynorphin/kappa system has not as in much of the cortex.4,8*12,17.22Kappa receptors been studied as extensively as in the rat. The dis- have been visualized via receptor autoradiography, tribution of prodynorphin-containing cells and with their distribution being similar to that of the fibers is similar in the primate, although the density prodynorphin-containing fibers.1’*‘2This would be of these cells is greater in primate cortex than in expected, given that prodynorphin is the only rodent cortex.3.7.‘0 Interestingly, the density of known source of endogenous kappa ligands. Pro- kappa receptors in the cortex also seems to increase dynorphin derived peptide concentrations have also with phylogeny, with cortical kappa receptors being been examined in dissected brain regions, using far more abundant in the primate than the radioimmunoassay. In general, the distribution of rodent.“,” Detailed examination of the specific immunoreactive prodynorphin derived peptides forms of prodynorphin-derived peptides expressed in primate brain, however, has not been performed. We undertook this study to begin to characterize Table 1 Cortical regions studied the expression of specific forms of prodynorphin in Region Corresponding selected brain regions of the primate. Given the studied cortical area?’ Function or common name relative abundance of prodynorphin fibers and cells, as well as IC receptors in the cortex of the 0 OB/OC visual TA TA primary auditory primate compared to the rodent, we have con- PG PG secondary association (IPL) centrated on the examination of the cortex, com- FD FD prefrontal paring it to the striatum. We have therefore L LA, LC cingulate FA FA motor determined the concentrations of 4 prodynorphin- A A temporal allocortex derived peptides, representing all three functional PB PB, PC primary somatosensory domains of the propeptide, in neocortical, allo- FB FB secondary motor (premotor) PE PE secondary somatosensory (area 5) cortical, and striatal regions of the old-world mon- key brain. PRODYNORPHIN-DERIVED PEPTIDE EXPRESSION IN PRIMATES 279 Methods licate. Radiolabeled peptides for RIA were pre- pared by chloramine-T-mediated iodination.6 Animals Antisera were raised in rabbits, and were the gift of The brains of 6 old world monkeys (Macaca neme- Dr Huda Akil (Mental Health Research Institute, strina) were obtained from several sources. All University of Michigan). monkeys were in good health and medication-free at the time of tissue acquisition. These monkeys Data analysis were ail sacrificed for other studies, and the brains Peptide concentration values from 3 independent were rapidly removed and frozen at - 80°C for later assays were averaged, and used for subsequent data study. analysis. Prodynorphin can be processed into three domains2 (Fig. 1). To estimate potential variations Tissue preparation of interdomain processing, the ratios of a-neo/[dyn Frozen whole brains were removed from - 80” stor- A (l-l 7) + dyn A (l-8) (hereafter, total dyn A)] and age and kept on wet ice until sufficiently thawed to dyn B/total dyn A were calculated. These ratios dissect (approximately - 20”). Brains were cut into would be expected to be unity if prodynorphin was 5-l 0 mm slabs in a coronal plane. These slabs were processed into equimolar amounts of the three conservatively dissected on wet ice into the specific domains, though because /I-neo and leumorphin regions of interest, using the atlas and nomenclature were not assayed, these ratios might be somewhat of Von Bonin and Bailey.” Dissected tissue was less than unity. To estimate possible processing promptly frozen on dry ice and stored at -80°C differences within one prodynorphin domain, the until extracted for assay. The individual regions ratio dyn A (l-l 7)/dyn A (l-8) was calculated. The were weighed while frozen, and immediately homo- dyn A region was chosen because dyn A (l-l 7) and genized in GT buffer (10 mM EDTA, 50 mM Tris dyn A (1-8) have different affinities for the opiate pH 7.5. and 5M guanidine thiocyanate) with a receptors, and processing differenees may thus be Brinkman Polytron. 6 volumes of 4M LiCl were important physiologically. Potential regional added to each sample, and samples were placed on differences were determined by analysis of variance, ice overnight. The samples were centrifuged for 30 with post-hoc Dunnett’s t-test. set at IO 000 rpm and the supernatant was trans- fered to a clean tube. Glacial acetic acid was added to a final volume of 5% and each sample was Results extracted using Sep-Pak C,, cartridges, as pre- Peptide content viously described.” The eluant from the cartridges was dried with a Savant Speed Vat rotary evap- The concentrations of the 4 prodynorphin derived orator. The dried extract was resuspended in meth- peptides were determined in the caudate, putamen, anol:O. 1 N HCl (1: 1, v:v), and stored at -20°C and 10 selected cortical regions, as shown in Figure until assayed. 2 and Table 2. For each peptide, the concentrations in the caudate and putamen were quite similar, although a-neo tended to be more abundant in the Radioimmunoassays putamen than in the caudate. a-neo was the most Radioimmunoassays (RIAs) were performed as abundant of the 4 peptides in both striatal regions, previously described.17.” Briefly, diluted tissue sam- which was 5-8 times the concentration of dyn B.