Steven R. Childers

Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat

Enkephalin immunoreactive perikarya, fibers and nerve terminals, visualized by the indirect immunohistofluorescent method in colchicine-pretreated animals, are localized in many discrete regions of the rat brain stem. These specific immunohistofluorescent patterns are similar after staining with selective primary antisera directed against either methionine-enkephalin or leucine-enkephalin. Cell bodies are found in the substantia gelatinosa and interpolaris zones of the trigeminal nuclear complex, the nucleus of the solitary tract, in the vicinity of the nucleus raphé magnus, in the dorsal cochlear, medial vestibular, and paraolivary nuclei and, dorsal to this last region, in the parabrachial nuclei and the dorsal tegmental nucleus of Gudden, in the periaqueductal gray matter and interpeduncular nucleus and along the borders of the lateral lemniscus and medial geniculate. In some areas, such as the parabrachial region, nucleus of the solitary tract and substantia gelatinosa of the trigeminal nucleus, these perikarya are associated with densities of fibers and terminals. Other regions, such as the dorsal cochlear nucleus and the vicinity of the nucleus raphé magnus, contain cell bodies associated with low densities of processes and terminals. In still other nuclei, such as the nucleus of the facial nerve and the locus coeruleus, fiber and terminal densities without associated cell bodies are evident. Many of these enkephalin localizations can be rationalized on the basis of known actions of opiate drugs and the brain stem distribution of opiate receptors.

Differential Regulation by Guanine Nucleotides of Opiate Agonist and Antagonist Receptor Interactions

Abstract: Guanine nucleotides differentiate binding of tritium‐labeled agonists and antagonists to rat brain membranes. In the absence of sodium, GTP (50 μM) decreased binding of [3H]‐labeled agonists by 20–60% and [3H]‐labeled antagonists by 0–20%. In the presence of 100 mM‐NaCl, GTP had no effect on antagonist binding, but decreased agonist binding by 60–95%. GMP was less potent than either GTP or GDP in decreasing agonist binding. GTP (50 μM) reduced high‐affinity [3H]dihydromorphine sites by 52% and low‐affinity sites by 55%. Without sodium, GTP reduced high‐affinity [3H]‐naloxone sites by 36%; in the presence of 100 mM‐NaCl, GTP had no effect on either high‐ or low‐affinity [3H]naloxone sites. GTP increased the association rate of [3H]dihydromorphine twofold and the dissociation rate by fourfold, while having no effect on association or dissociation rates of the antagonist [3H]diprenorphine. The affinities of uniabeled antagonists in inhibiting [3H]‐diprenorphine binding were not affected by GTP or sodium, but the affinities of agonists were reduced 40‐ 120‐fold, with met‐ and leu‐enkephalin affinities reduced by the greatest degree. GTP and sodium lowered [3H]dihydromorphine binding in an additive fashion, while divalent cations, especially manganese, reversed the effects of GTP on [3H]‐labeled agonist binding by stimulating membrane‐bound phosphatases that hydrolyze GTP to GMP and guanosine. These results suggest that by affecting binding of agonists, but not antagonists, GTP may regulate opiate receptor interactions with their physiological effectors.

Guanine nucleotides differentiate agonist and antagonist interactions with opiate receptors

Abstract Opiate receptor binding is regulated by guanine nucleotides differentially for agonists and antagonists. Guanosine-5′-triphosphate (GTP), its stable analogue guanyl-5′-yl-imidodiphosphate (Gpp(NH)p) and GDP inhibit binding of the 3H-agonists dihydromorphine, etorphine and enkephalins but not the 3H-antagonists naloxone or diprenorphine. GMP, ATP, ADP and AMP fail to alter either agonist or antagonist binding. Effects are more pronounced in the presence than in the absence of sodium.

The opiate receptor binding interactions of 3H-methionine enkephalin, an opioid peptide

3H-Methionine enkephalin binds stereospecifically with high affinity to opiate receptors in rat brain membranes. Equilibrium experiments indicate two distinct dissociation constants with KD values of 1.8 and 5.8 nM respectively. 3H-Methionine enkephalin associates and dissociates from the opiate receptor with 8--10 fold slower kinetics than 3H-opiates. Though several opiates have similar affinities for sites labeled by 3H-methionine enkephalin, 3H-dihydromorphine and 3H-naloxone, some opiates such as morphine, dihydromorphine and oxymorphone are only one tenth as potent in competing for 3H-methionine enkephalin as for 3H-dihydromorphine and 3H-naloxone binding. As with other opiate agonists, 5--10 mM sodium selectively decreases the binding of 3H-methionine enkephalin. At 26 degrees C, 0.1--1.0 mM manganese but not magnesium or calcium increases the binding of 3H-methionine enkephalin, while at 0 degrees C manganese decreases the binding of methionine enkephalin.

Characterization of [3H]Guanine Nucleotide Binding Sites in Brain Membranes

Abstract: [3H]GTP [guanosine triphosphate] and [3H]GMP‐PNP [guanosine 5′‐(β,8‐imino)triphosphate, a nonmetabolized analog of GTP] have been utilized as ligands to characterize binding sites of guanine nucleotides to rat brain membranes. Binding of both [3H]GTP and [3H]GMP‐PNP is saturable, with respective KD values of 0.76 and 0.42 μM. The number of binding sites for GMP‐PNP (4 nmol/g) is three times greater than for GTP (1.5 nmol/g). This discrepancy is caused by rapid degradation of GTP to guanosine by brain membranes, which can be partially prevented by addition of 100 μM‐ATP. The binding of [3H]guanine nucleotides is selective, with approximately equipotent inhibition by GTP, GDP, and GMP‐PNP (at 0.2‐1.0 μM), but no inhibition by other nucleotides at 100 μM concentrations. The binding sites for guanine nucleotides in brain membranes appear not to be associated with microtubules, since treatments that reduce [3H]colchicine binding by 65% have no effect on [3H]GTP binding. [3H]Guanine nucleotide binding is widely distributed in various organs, with highest levels in liver and brain and lowest levels in skeletal muscle. The characteristics of these binding sites in brain show specificity properties of sites that regulate neurotransmitter receptors and adenylate cy‐clase.

Enkephalin levels in mouse brain: diurnal variation, post-mortem degradation, and effect of cycloheximide

Enkephalin was first isolated as a mixture of the two pentapeptides methionine enkephalin (met-enkephalin) and leucine enkephalin (leu-enkephalin) by its ability to inhibit electrically induced contractions of the guinea pig ileum (Hughes, 1975; Hughes et al., 1976) and to compete with [3H] opiate binding to rat brain membranes (Terenius and Wahlstrom, 1974; Pasternak et al., 1975; Simantov and Snyder, 1976a). Although these assays have been useful in elucidating several properties of opioid peptides such as their regional (Hughes, 1975; Pasternak et al., 1975) and subcellular distributions (Simantov and Snyder, 1976b), their lack of specificity makes it very difficult to distinguish the various opioid peptides from each other. Specific radioimmunoassays (RIA) for met and leu-enkephalin (Simantov et al., 1977; Yang et al., 1977) not only distinguish met and leu-enkephalin but also discriminate between enkephalin and the larger endorphin fragments of β-lipotropin (β-LPH). In this way enkephalin RIAs can detect changes in enkephalin levels which result from specific enkephalinergic metabolic processes in the brain.

Differential binding properties of some opiates and opioid peptides

The opioid peptide enkephalins were discovered as endogenous substances in the brain which mimic effects of morphine pharmacologically at presumed receptor sites in smooth muscle systems (Hughes, 1975) and compete for [3H] opiate binding to opiate receptor sites in brain membranes (Terenius and Wahlstrom, 1975; Pasternak et al. 1975a). Accordingly, these opiate receptor sites which bind opiates with high affinity and in proportion to their pharmacological activity (Snyder, 1975) presumably serve physiologically to interact with endogenous opioid peptides. In the brain the quantitatively predominant opioid peptides are the two pentapeptides met and leu-enkephalin while lower concentrations of a 39-amino acid opioid peptide β-endorphin may also presumably interact with opiate receptors. However, since the localization of enkephalin throughout the brain corresponds more closely with that of opiate receptors than does localization of β-endorphin (Simantov et al. 1977; Rossier et al. 1977), it is likely that the majority of opiate receptors in the brain normally interact with enkephalins rather than β-endorphin.

CHARACTERIZATION OF HIGH MOLECULAR WEIGHT ENKEPHALIN IMMUNOREACTIVITY IN RAT BRAIN AND BOVINE ADRENAL MEDULLA

Summary Assay of high molecular weight soluble proteins in rat brain revealed the presence of two peaks of enkephalin-like immunoreactivity: one (>100,000 MW) non-specifically interacted with immunoglobulins, and a second (MW ~ 40,000) reacted in both met-and leu-enkephalin radioimmunoassays. Incubation of the second peak with trypsin resulted in the formation of enkephalin-like immunoreactivity of MW approx. 1000 and loss of activity in the 40,000 MW peak. These results suggest that enkephalin in brain may be formed by breakdown of large precursors by non-trypsin proteases. Analysis of enkephalin-like immunoreactivity in bovine adrenal medulla chromaffin granules reveals two different peaks, MW approx. 30,000 and MW approx. 600-1000. Preliminary results suggest that these adrenal components differ from the brain 40,000 MW activity and with enkephalin itself.

Opiate Receptors: A) Functional Heterogeneity Demonstrated with an Apparently Irreversible Naloxone Derivative: B) Regulation by Guanine Nucleotides

ABSTRACT 3H-Opiate binding to receptor sites can be resolved into separate high and low affinity sites. Only the high affinity 3H-agonist site is affected by sodium, which essentially abolishes it. Naloxazine, an hydrazine derivative of naloxone, lowers opiate receptor binding markedly in extensively washed membranes up to 24 hrs following its administration to intact animals. Naloxazine administration in vivo abolishes high affinity binding with no influence on low affinity sites. Sixteen hours after its in vivo administration, naloxazine still prevents the analgesic effects of morphine at a time when effects of naloxone have dissipated. These findings indicate that high affinity sites are responsible for these pharmacological opiate actions. Opiate agonist binding is inhibited selectively by guanine nucleotides. While GTP, Gpp(NH)p and GDP are effective, GMP and adenine nucleotides are ineffective. The influence of guanine nucleotides is manifested with several opiate agonists but not with antagonists.