Dehydro-enkephalins. IV. Discriminative recognition of delta and mu opiate receptors by enkephalin analogs

We have studied the receptor binding activities of $\text{C}$-terminal free and amidated enkephalins with and without the dehydrophenylalanine⁴ residue. For the selective labeling of so-called δ and μ opiate receptors, specific tracers were used at low concentrations in rat brain membranes and neuroblastoma cells containing pure δ receptors. $\text{C}$-Terminal free enkephalins are five to eight times more potent in the assay for δ receptors than in μ assay, while the amides are almost equipotent in both assays. The presence of a C-terminal carboxyl group is a determining factor for selective activity. [D-Ala², ΔPhe⁴, Met⁵]-enkephalin amide is very potent in all of the binding assays examined, and, in particular, twice as active as the saturated amide and the $\text{C}$-terminal free enkephalin in the δ assay. We suggest that the steric arrangement of the dehydrophenylalanine residue in position 4 is very important to the enhanced interaction with the δ receptors.     

Fluorescence decay time distribution analysis of cyclic enkephalin analogues; influence of solvent and Leu configuration in position 5 on conformation.

Lifetime distribution analysis were performed to study the influence of Leu configuration in position 5 on changes of the peptide chain of cyclic analogues of enkephalins containing a fluorescence donor and acceptor in different solvents. The configuration change of Leu5 in all the analogues of enkephalins studied which contain donor-acceptor pairs has no apparent influence on Trp lifetime distributions. In contrast, there is a significant solvent effect on the shape of lifetime distribution.     

Reserpine Increases Chromaffin Cell Enkephalin Stores Without a Concomitant Decrease in Other Proenkephalin-Derived Peptides

Reserpine increases the levels of enkephalins in adrenal medullary chromaffin cells; however, the origin of the newly apparent pentapeptides has been the subject of debate, because no increase in the levels of proenkephalin mRNA has been observed. The present study was performed for determining if the reserpine-induced increase in context of enkephalins was derived from processing of preexisting fragments of proenkephalin. Bovine chromaffin cell enkephalins and larger enkephalin-containing peptides were separated by reversed-phase HPLC and identified by approximate molecular weight, elution with peptide standards, and enkephalin sequences contained. Treatment of the cells with reserpine increased the levels of enkephalins and of enkephalin-containing peptides of up to approximately 3 kilo-daltons without reducing the levels of larger enkephalin-containing peptides. Similar results were obtained with another catecholamine-depleting drug, tetrabenazine. In contrast, treatment of chromaffin cells with theophylline or forskolin increased the levels of both enkephalins and enkephalin-containing peptides of all sizes. The results suggest that new synthesis of proenkephalin is required for the effects of reserpine, although proenkephalin processing is also altered by this drug.     

Motilin and a synthetic enkephalin induce colonic motor complexes (CMC) in the conscious dog

In the conscious dog migrating and nonmigrating colonic motor complexes (CMC) were recorded by means of chronically implanted strain gauge force transducers. Intravenous injection of a synthetic enkephalin analogue immediately induced a premature CMC at all three recording sites of the colon. Naloxone inhibited the enkephalin- but not motilin-induced CMC. We therefore exclude that motilin acts by release of endogenous enkephalins. The two peptides stimulate CMC in the canine colon by different pathways. Naloxone alone had no effect on normal colonic motility, suggesting strongly that endogenous enkephalins do not modulate regular CMC in the dog.     

Enkephalin-binding systems in human plasma

Three amino acid-containing fractions present in human plasma are shown to bind both leu and met-enkephalin: serum albumin and two species of a much lower molecular weight, in all likelihood polypeptides. The amount of enkephalin associated with serum albumin seems comparatively smaller than that associated with the two low molecular weight systems. These systems jointly are apparently capable of binding a significant part of the circulating enkephalins. The possibility is suggested that the interactions described may play a role in maintaining the integrity of circulating enkephalins.     

Tetraethylammonium facilitates the stimulation-evoked loss of the enkephalins from the myenteric plexus of guinea-pig ileum

The effects of electrical field stimulation on the contents of [Met]enkephalin and [Leu]enkephalin were determined in myenteric plexus-longitudinal muscle preparations of the guinea-pig small intestine. Cycloheximide (0.1 mM) was present in all experiments to prevent de nouveau biosynthesis. The two enkephalins were separated by high performance liquid chromatography and assayed on the mouse vas deferens. Stimulation with submaximal pulses (50 mA, 0.5 ms) at a frequency of 10 Hz caused maximal losses of about 35% of [Met]enkephalin and [Leu]enkephalin after 3 h (108000 pulses). The plot of log (enkephalin content) against number of pulses was steeper during the first 30 min than during the later periods. Tetraethylammonium bromide (TEA, 10 mM) increased the [Met]enkephalin and [Leu]enkephalin contents of the non-stimulated preparations by about 50%. When the preparations were stimulated in the presence of TEA at 50 mA and 1 Hz, the plots of loss of enkephalins against number of pulses were linear until the maximum of about 50% was reached. Compared with stimulation in the absence of TEA, the rate constant was 8 times greater for [Leu]enkephalin and 20 times greater for [Met]enkephalin. The absolute losses per pulse were about 13 times greater for [Leu]enkephalin and 27 times greater for [Met]enkephalin than in the absence of TEA. In the presence of bacitracin and a mixture of dipeptides, the enzymatic degradation of the enkephalins was sufficiently suppressed to cause an overflow of 30-60% of the enkephalins lost from their stores into the perifusing Krebs solution. Until it is possible to determine the preformed precursors, which are present in large quantities, the kinetics relationship between these precursors and the enkephalins cannot be investigated. A similar dilemma exists for the relationship between "released' enkephalins and the losses from their stores.     

Are NPY and enkephalins costored in the same noradrenergic neurons and vesicles?

Separate studies show that NPY and enkephalins are widely distributed in peripheral noradrenergic neurons. In the present study, the subcellular costorage and release in response to intense sympathetic stimulation and reserpine at near therapeutic doses (0.05 mg/kg every other day) were examined. In young pig arteries and vas deferens, enkephalin and D beta H immunofluorescence show consistent but not total overlap. Also NPY is colocalized with D beta H in many fibers but with VIP (nonnoradrenergic) in others. Ultrastructural immunogold labeling indicates that individual terminals contain large dense cored vesicles (LDVs) which store either NPY or enkephalins, even though costorage of both peptides occurs. Some LDVs costore NPY and VIP, especially in the middle cerebral artery and in the lamina propria of vas deferens. Acute CNS ischemia depletes enkephalins and norepinephrine in all tissues analyzed without parallel loss of NPY. Reserpine depletes norepinephrine 70-85% but does not deplete NPY or enkephalins. The latter is in contrast to commonly used high doses known to produce nonspecific, detergent-like effects. In fact, low doses of reserpine induce a time-dependent new synthesis and processing of NPY precursor peptides in vas deferns. Contrasting effects of reserpine on NPY and enkephalin contents, new synthesis and apparent processing, and a differential response to acute CNS ischemia were found in every tissue studied. Activation of precursor neuropeptide processing occurred immediately upon intense sympathetic stimulation in most tissues. Dual localization of NPY in noradrenergic and nonnoradrenergic fibers and differences in subcellular LDV storage help explain why enkephalin correlates better than NPY with norepinephrine loss in response to acute CNS ischemia. Furthermore, the costorage of NPY and enkephalins in distinct subpopulations of noradrenergic fibers, which varies according to tissue, is likely to be under separate CNS control.     

Synthesis of enkephalins by guinea-pig striatum in vitro.

A method for obtaining incorporation of labelled amino acids into enkephalins of guinea-pig striatum in vitro and isolating the labelled enkephalins is described. The incorporation of [3H]tyrosine increases with time after a delay of 2 h. Cycloheximide added during this time, but not at later stages, inhibits incorporation. The results indicate that the enkephalins are produced locally, probably by ribosomal synthesis. The lag period may be due to the time required for the synthesis of precursors and their conversion to the enkephalins.     

Catatonic or hypotonic immobility induced in mice by intracerebroventricular injection of mu or kappa opioid receptor agonists as well as enkephalins or inhibitors of their degradation

The intracerebroventricular injections in mice of the mu receptor agonists morphine and fentanyl induced an immobility state (the animals staying motionless with the head down on a 45° inclined plane) which was apparently hypertonic (catatonia ?) or at least enabled them to remain hanging on a horizontal wire with their forepaws. In similar conditions, injections of the kappa receptor agonists ketocyclazocine and bremazocine induced an immobility state which was hypotonic, in contrast with the preceding one. In a similar way to the mu agonists, Met-enkephalin or Leu-enkephalin injected i.c.v. in association with the inhibitor of enkephalinase thiorphan induced an apparently hypertonic immobility which was easily antagonized by naloxone. The association of thiorphan with bestatin ( an inhibitor of aminopeptidases involved in enkephalins inactivation ) produced similar results. In contrast, the hypotonic immobility induced by the kappa receptor agonists required relatively high doses of naloxone to be antagonized. The opiate antagonist MR 2266 antagonized equipotent doses of all the above mentioned agents with a similar efficacy. From these data it is suggested that enkephalins could induce an apparently tonic immobility by stimulating mu receptors and that endogenous enkephalins could be involved in a tonic mediation modulating the locomotor activity or regulating the muscular tone.     

Fluorescent enkephalin derivatives with biological activity

Fluorescent dansylated derivatives of enkephalins were prepared, with the dansyl group either at the N- or C-terminal part of methionine-enkephalin (Met-E) or its peptidase-resistant analogue D-Ala₂-Met-E. An energy transfer, from the Tyr to the dansyl group, was found for all compounds and spectral properties were dependent upon the environment polarity. Met-E-(CH₂)₂ dansyl II and D-Ala₂-Met-E-(CH₂)₂ dansyl III exhibit biological activities on guinea-pig ileum and binding affinities on striatal membranes, similar to Met-E. III exhibits significant analgesic activity in mice after i.v. administration (about 35 % that of morphine). Fluorescent enkephalins potentially represent useful probes for the exploration of opiate receptors, studies of ligand-receptor interactions and evaluation of enkephalins cleaving peptidases in various tissues.     

Enkephalins and hormonal-metabolic reactions in experimental stress of differing severity

Experiments on rats were made to study the effects of enkephalins on blood cortisol, insulin and glucose and excretion of catecholamines with urine during stress. In mild stress, enkephalins were demonstrated to be able to increase the catabolic processes, thereby accelerating the transition of the reaction to the phase of resistance. In severe stress, parenteral administration of enkephalins was discovered to promote the reduction in alterations of the parameters under study characteristic of stress.     

Postsynaptic inhibitory effects of Met- and Leu-enkephalin on endocrine and adjacent neurones in the preoptic-septal region of the guinea pig

Iontophoretic application of enkephalins induced inhibitory effects on unit activity of endocrine and adjacent neurones in the preoptic-septal region. Antagonism or lack of antagonism of these effects by naloxone indicated an action of enkephalins through different opiate receptors. Inhibitory effects of enkephalins were obtained during iontophoretic application of Mg2+, showing that these opioid peptides acted postsynaptically. Because enkephalin acts on endocrine neurones, these opioid peptides might be involved in the control of gonadotrophic hormone release by acting on cell bodies of LH-RH neurones.     

Selective inhibition of synaptosomal proline uptake by leucine and methionine enkephalins

The high affinity, sodium-dependent uptake of proline by rat brain synaptosomes was inhibited by the opioid pentapeptides, Leu-enkephalin and Met-enkephalin. The synaptosomal uptake of other putative neurotransmitter amino acids including glutamic acid, aspartic acid, gamma-aminobutyric acid, and taurine was not altered in the presence of enkephalins. The uptake of a neuroinactive amino acid, leucine, was also unaffected by enkephalins. The extent of proline uptake was half-maximal at a Leu-enkephalin concentration of 1 microM. Both the initial rate of transport and the overall capacity for proline accumulation were reduced. The effect of the enkephalins was vectorial since carrier-mediated efflux of proline was not altered in the presence of enkephalins. Morphine and the opioid peptides, dynorphin and beta-endorphin, were without effect on proline uptake. The inhibition of proline uptake by enkephalins was not diminished by prior incubation of the synaptosomal preparation with naloxone; however, the inhibition was attenuated by 1-butanol. The des-tyrosyl fragments of the enkephalins were as inhibitory as the intact pentapeptides. A modified enkephalin ([D-Ser2]Leu-enkephalin-Thr) with selective affinity for the delta subclass of enkephalin receptor was effective in inhibiting proline uptake. On the basis of the selectivity of these effects, we propose that there is a specific population of nerve endings in the cerebral cortex that contains both a proline-transport system and binding sites for Leu- and Met-enkephalin and furthermore, that these binding sites may be related to the putative delta receptor.     

Structural alterations of enkephalins in the presence of GM1 ganglioside micelles

The enkephalins are endogenous neurotransmitters and bind with high affinity at the delta-receptor. Gangliosides, the major glycans of nerve cells, known to interact both with receptors and ligands on the cell surface, have been implicated to modulate the actions of opioid receptors by allosteric regulation (Wu, G.; Lu, Z. H.; Wei, T. J.; Howells, R. D.; Christoffers, K.; Leeden R. W. Ann NY Acad Sci 1998, 845, 126-138). We have studied the interactions between enkephalins and monosialylated ganglioside GM1 using NMR spectroscopy and fluorescence. The structural models of enkephalins in the presence of GM1 micelles were generated using two-dimensional (1)H-ROESY experiments along with restrained molecular dynamics simulations. We report a conformational alteration of enkephalins in the presence of GM1 micelles.     

Hydrolysis and protection from hydrolysis of enkephalins in human plasma

Seven groups of enkephalin-degrading enzymes and three groups of inhibitors active on these enzymes were separated from human plasma. The activity of the enzymes in hydrolyzing enkephalins and of the inhibitors in protecting enkephalins from proteolysis was measured. Results obtained with the endogenous inhibitors were compared to those relative to synthetic inhibitors. Data obtained indicate that all enkephalin-degrading enzymes found in plasma are significantly inhibited by the endogenous substances present in this tissue. The inhibition of the different classes of plasma enzymes by two of the three groups of endogenous substances is quite uniform, while one group of inhibitors appears specific to dipeptidylpeptidases. Results obtained are discussed in terms of the functional role of the inhibitory substances and of the possible pharmacological implication of their presence in human plasma.     

The peroxidase-catalyzed oxidation of enkephalins.

In vitro experiments are reported showing that Leu-enkephalin and Metenkephalin, in the presence of hydrogen peroxide, can be oxidized by horseradish peroxidase. The products formed are strongly fluorescent and characterized by absorption peaks with maxima at 290 nm and 315 nm. The effects of substrate and enzyme concentrations on the oxidation rate of enkephalins are described. Amino acid analysis of the hydrolysates from peroxidase-treated enkephalins provides evidence for the presence of dityrosine. The data suggest that the oxidation leads to the production of enkephalin dimers with a linkage between the N-terminal tyrosine residues. Data are also obtained indicating that enkephalins function as hydrogen donors for mammalian peroxidases.     

Identification of aldosterone secretion inhibitory factor in bovine adrenal medulla

We report the first demonstration of an Aldosterone Secretion Inhibitory Factor (ASIF) in acid extracts of bovine adrenal medulla. Following separation from catecholamines and enkephalins, this factor leads to an 80% inhibition of PGE1-stimulated secretion of aldosterone from bovine adrenal zona glomerulosa. ASIF is retained on cation exchange gels and behaves as a small 5K-dalton peptide on Sephadex G-50. This factor cross-reacts in a radio-receptor assay for [125I] atrial natriuretic factor (ANF). ASIF is distinct from all neuropeptides formerly detected in the adrenal medulla, e.g. somatostatin, enkephalin, neuropeptide Y, dynorphin, neurotensin. In the adrenal gland, this ANF-like factor is predominantly found in the medulla (4 pmol/mg protein), with only trace amounts in the cortex (0.1 pmol/mg protein). ASIF might perhaps correspond to the endogenous ligand for the receptor sites that we have previously identified with [125I]ANF in bovine adrenal cortex and could contribute to the formerly reported attenuating influence of the adrenal medulla on mineralocorticoid production.     

Influence of enkephalins on the ACTH-induced hormonal imprinting in tetrahymena

Both adrenocorticotrop hormone (ACTH) and the synthetic enkephalins investigated evoked imprinting in Tetrahymena and led to increased hormone binding at further contact with ACTH. Neither molecule evoked, however, imprinting for the enkephalins. The pentapeptide enkephalin containing also proline had the most pronounced imprinting effect and, when given together with ACTH, it increased the imprintatory effect of ACTH considerably. In all the situations investigated the enkephalin tetrapeptide inhibited the positive effect of the enkephalin pentapeptide, whereas it did not influence the imprintatory effect of ACTH. Similarities can be found between the pharmacological and imprinting effects of enkephalin in mammals, and the effects seen in the present investigations.     

Proenkephalin expression and enkephalin release are widely observed in non-neuronal tissues

Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. Studies have shown that enkephalins play an important role in behavior, pain, cardiac function, cellular growth, immunity, and ischemic tolerance. Our global hypothesis is that enkephalins are released from non-neuronal tissues in response to brief ischemia or exercise, and that this release contributes to cardioprotection. To identify tissues that could serve as potential sources of enkephalins, we used real-time PCR, Western blot analysis, ELISA, immunofluorescence microscopy, and ex vivo models of enkephalin release. We found widespread expression of preproenkephalin (pPENK) mRNA and production of the enkephalin precursor protein proenkephalin (PENK) in rat and mouse tissues, as well as in tissues and cells from humans and pigs. Immunofluorescence microscopy with anti-enkephalin antisera demonstrated immunoreactivity in rat tissues, including heart and skeletal muscle myocytes, intestinal and kidney epithelium, and intestinal smooth muscle cells. Finally, isolated tissue studies showed that heart, skeletal muscle, and intestine released enkephalins ex vivo. Together our studies indicate that multiple non-neuronal tissues produce PENK and release enkephalins. These data support the hypothesis that non-neuronal tissues could play a role in both local and systemic enkephalin-mediated effects.     

Mu and delta receptors: their role in analgesia in the differential effects of opioid peptides on analgesia

Utilizing the mouse tail-flick assay, the rank order of analgesic potency for various opioids (i.c.v.) is beta h-endorphin greater than D-Ala2-D-Leu5-enkephalin greater than morphine greater than D-Ala2-met-enkephalinamide much greater than met-enkephalin much greater than leu-enkephalin. Assuming mu receptor mediation of analgesia, there is an affinity and analgesic potency (ie: D-Ala2-Leu5-enkephalin has 1/7 the affinity of morphine for the mu receptor but is 18X more potent as an analgesic). Additionally, sub-analgesic doses of various opioid peptides have opposite effects on analgesic responses. Leu-enkephalin, D-Ala2-D-Leu5-enkephalin or beta h-endorphin potentiate morphine or D-Ala2-met-enkephalinamide analgesia whereas met-enkephalin or D-Ala2-met-enkephalinamide antagonize opioid-induced analgesia. Using the enkephalins as the prototypic delta ligands (100 fold selective) and based on their effects on analgesia, we suggest that Leu-enkephalin-like peptides interact with the delta receptor as an "agonist" to facilitate and met-enkephalin-like peptides as an "antagonist" to attenuate analgesia. Given the biochemical evidence of a coupling between mu and delta receptors, we suggest that the mechanism of facilitation or attenuation of analgesia by the enkephalins is a direct in vivo consequence of this coupling. Further, the analgesic potencies of various opioid ligands can be better correlated to the combination of their simultaneous occupancy of mu and delta receptors.