Pulse-chase studies of the POMC/Beta-endorphin system in the pituitary of acutely and chronically stressed rats

Experiments were carried out to determine whether stress induces biochemical changes in the pro-opiomelanocortin (POMC) system in anterior (AL) and intermediate-posterior lobe (IPL) of rat. In a series of pulse-chase experiments, acute stress led to an increase in POMC biosynthesis and shorter half-life in AL. However, when the animals were chronically stressed, the AL no longer exhibited increased POMC synthesis. On the other hand, in the IPL, acute stress did not produce any biochemical changes, but chronic stress led to an increase in POMC synthesis and shorter half-life. These data suggest that AL and IPL are affected by acute and/or chronic exposure to stress in opposite directions and that the POMC system in AL may play an important role in stress-induced analgesia.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23803/1/0000041.pd

Dynorphin is located throughout the CNS and is often co-localized with alpha-neo-endorphin

The opioid peptide dynorphin has been described as widely distributed in CNS when measured by RIA. Our previous immunohistochemical studies have only demonstrated dynorphin cells as those containing AVP. We now report the specific localization of dynorphin throughout the neuraxis. Further, dynorphin and alpha-neo-endorphin have been co-localized to the same magnocellular neurosecretory cells in hypothalamus. We report agreement with the findings of others and extend them to include a cell group in dorsomedial hypothalmus, further strengthening the association between dynorphin and alpha-neo-endorphin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23834/1/0000073.pd

The signal peptide of pro-opiomelancortin: Validation of a specific radioimmunoassay

The N-terminus portion of the POMC leader sequence (signal peptide) was synthesized, and an antiserum was raised against it. A radioimmunoassay was developed which is effective at a dilution of 1:500,000, and sensitive at less than 1 fmole/tube. Since leader sequences often exhibit structural homologies, and since synthetic peptides are not readily available, we resorted to an unusual procedure to establish specificity. This involved extraction of pituitary RNA, cell-free translation to produce the pre-prohormones, and purification by B-END and signal antibody affinity columns. The eluates were then tested by SDS gel electrophoresis and by multiple immunoprecipitations. All results showed that the signal antibody captured a single molecular species, approximately 30,000 in MW, which was also captured by the B-END column, and was immunoprecipitable by B-END and ACTH antisera. It therefore appears that this antibody selectively measures the POMC leader sequence and should be valuable in measuring the newly synthesized pre-prohormone.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23804/1/0000042.pd

Des-tyrosine-dynorphin antagonizes morphine analgesia

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23809/1/0000047.pd

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300-400 [mu]g) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1-13, 7-17, or 1-17). For comparison, antisera to [Leu]enkephalin (residues 1-5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23823/1/0000062.pd

Site-directed mutagenesis of the human dopamine D2 receptor

Based on amino acid sequence and computer modeling, two conflicting three-dimensional models of the dopamine D2 receptor have been proposed. One model (Dahl et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8111) suggests that dopamine interacts with aspartate 80 of transmembrane (TM) 2 and asparagine 390 of TM6 with the transmembranes arranged in a clockwise manner, while a second model (Hibert et al., 1991, Mol. Pharmacol. 40, 8) suggests that dopamine interacts with aspartate 114 of TM3 and the serines of TM5 (194 and 197) with the transmembranes arranged in a counterclockwise manner when viewed from the extracellular space. The present study tests the latter model by selectively mutating aspartate 114 and serines 194 and 197 of the human dopamine D2 receptor by site-directed mutagenesis. In addition, two methionines (116 and 117) were mutated to evaluate whether residues near aspartate (114) of the dopamine D2 receptor are critical in differentiating dopamine receptor agonists from adrenoceptor agonists. Removal of the negative charge with the mutation of aspartate (114) to either asparagine or glycine led to a total loss of both agonist and antagonist binding. Individual or dual methionine mutations in positions 116 and 117, to make the dopamine D2 binding pocket more closely resemble the [beta]2-adrenoceptor, did not result in a change in selectivity toward noradrenergic agonists or antagonists. The serine mutations revealed interesting differences between the dopamine D2 receptor and the adrenoceptors. In particular serine 197 appeared more important than serine 194 for agonist binding. In addition, the binding of one agonist (N-0437) was unaffected by individual serine mutations, while the binding of some antagonists, such as raclopride and spiperone, was significantly altered. These findings are discussed in relation to ligand structure and their interactions with the putative binding pocket.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29803/1/0000149.pd

A comparison of D1 receptor binding and mRNA in rat brain using receptor autoradiographic and in situ hybridization techniques

D1, a subtype of the dopamine receptors, is widely distributed in the nervous system and has been shown to be positively coupled to adenylate cyclase. Using a combination of in vitro receptor autoradiographic and in situ hybridization techniques, the present study examines the co-distribution of D1 receptor binding sites and D1 receptor messenger RNA in adjacent rat brain sections. D1 receptor binding sites were labeled using the selective antagonist [3H]SCH23390 (4.6 nM) in the presence of 1 [mu]M ketanserin, while the D1 receptor messenger RNA was visualized with a 35S-labeled riboprobe corresponding to a region between transmembrane domains III and VI of the rat D1 receptor (bp 383-843). Analysis of serial sections suggested a good agreement between D1 receptor binding and messenger RNA in several brain regions, including the paleocortex, caudate-putamen, nucleus accumbens, amygdala and suprachiasmatic nucleus. Marked discrepancies between D1 receptor binding and messenger RNA were observed in other brain regions including the entopeduncular and subthalamic nuclei, substantia nigra (pars reticulata), hippocampus and cerebellum. While technical considerations may contribute to these results, much of the discordance between the distributions is likely due to the differential localization D1 receptor messenger RNA in cell bodies and receptor binding sites on fibers and may provide insights into receptor synthesis, transport and membrane insertion. In the basal ganglia, for instance, D1 receptors are synthesized in the striatum and are either transported to efferent projections in areas such as the substantia nigra, or remain localized in striatal cells bodies. Ibotenic acid lesions in the striatum are consistent with these conclusions and demonstrate a coordinate loss of D1 receptor binding and messenger RNA in the caudate-putamen that is accompanied by a degeneration of fibers projecting to substantia nigra and a loss of D1 binding in the pars reticulata. Neurons in the dentate gyrus and in the granular layer of the cerebellum, on the other hand, synthesize D1 receptors and transport them entirely to either their dendritic or axonal fields, respectively, in the molecular layer.This analysis provides a better understanding of dopaminergic receptor systems in the CNS and their anatomical organization.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29608/1/0000697.pd

Dynorphin (1-17): Lack of analgesia but evidence for non-opiate electrophysiological and motor effects

Dynorphin and an opiate-inactive fragment des-Tyr-dynorphin produced similar effects on EEG, motor function and hippocampal unit firing. Naloxone had no effect on the actions of dynorphin in these systems and dynorphin failed to produce analgesia upon central administration. These results suggest that dynorphin has a pharmacological character that differs significantly from the classic narcotics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23835/1/0000074.pd

[3H]dynorphin a binding and [kappa] selectivity of prodynorphin peptides in rat, guinea-pig and monkey brain

We have previously demonstrated that [3H]dynorphin A selectively labels [kappa] opioid receptors in guinea-pig whole brain. In these current studies, using protection from inactivation by [beta]-chloronaltrexamine ([beta]-CNA), we are able to demonstrate that although dynorphin A prefers [kappa] receptors, it will label [mu] receptors when [kappa] receptors are not available, or present in only a small number. Thus, differences in numbers of [mu] and [kappa] receptors present in brain preparations are critical in determining the receptor binding profile of [3H]dynorphin A across species. Additionally, lthough all the prodynorphin derived peptides show [kappa] preference, the ability of the other prodynorphin derived peptides to compete with [3H]dynorphin A for its receptor varies across species. Consequently, in a highly enriched [kappa] preparation such as monkey cerebral cortex, [3H]dynorphin A appears to label [kappa] receptors with substantial selectivity, and the other prodynorphin-derived peptides show less ability to compete with dynorphin A for its receptor. In contrast, in a [kappa]-poor tissue such as rat brain, all of the prodynorphin-derived peptides, including dynorphin A-(1-8), show very similar potency. Thus, differences in [mu] and [kappa] receptor numbers across brain regions and species lead to differences in the receptor binding profile of dynorphin A.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26234/1/0000314.pd

Regional processing of the N- and C-terminal domains of proopiomelanocortin in monkey pituitary and brain

The total content and extent of processing of the [gamma]3MSH and [beta]-endorphin-containing N- and C-terminal domains of proopiomelanocortin were determined in the anterior and intermediate lobes of the pituitaries and in 11 regions of the brains of three Rhesus monkeys. Most immunoreactive [gamma]3MSH and [beta]-endorphin was located in the pituitary lobes, although significant amounts were also found in several brain regions. Sephadex column chromatography revealed that [gamma]3MSH immunoreactivity was found primarily as 4K and 9K forms; no [gamma]1MSH was detected. [beta]-Endorphin immunoreactivity was found as [beta]-endorphin, [beta]-lipotropin, and as a 5K form which may represent [beta]-endorphin extended N-terminally by part or all of [beta]-MSH. In the anterior lobe of the pituitary, the predominant products were 9K [gamma]3MSH and [beta]-lipotropin; in the intermediate lobe, more processed forms (4K [gamma]3MSH, [beta]-endorphin and 5K [beta]-endorphin) appeared to be preferentially stored. The pattern of processing in various brain regions was similar to that of the intermediate lobe of the pituitary.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27343/1/0000368.pd