Enkephalin Peptides Research Articles

Optimization of the conditions for biuret complex formation for the determination of peptides by capillary electrophoresis with ultraviolet detection

Capillary electrophoresis with UV detection was utilized to optimize copper complexation conditions for the analysis of neuropeptides. Complexation was confirmed by monitoring the response at a visible wavelength. Four complexation strategies were used to compare the UV response of native peptides and their respective copper complexes. All four strategies resulted in complete complexation, but on-capillary complexation provided significant advantages over precapillary and pre-/on-capillary. An increase in UV absorbance along with peak stacking resulted in a significantly greater response using the on-capillary technique. Also, on-capillary complexation does not require dilution of the sample. The effects of temperature and copper concentration were also investigated. The utility of this method for the separation of an enkephalin peptide mixture is presented.

Expression and development of the proenkephalin mRNA in the C cells of chicken ultimobranchial glands

A large number of enkephalin-immunoreactive cells transiently appear in chick ultimobranchial glands during embryonic development. The expression and development of proenkephalin mRNA were examined in the ultimobranchial glands by in situ hybridization with digoxigenin (DIG)-labeled oligonucleotide probes, in comparison with those of calcitonin mRNA and enkephalin peptide. Proenkephalin mRNA, as well as calcitonin mRNA, appeared in some C cells at embryonal day 14 (E 14), and in many cells at E 16. Subsequently, there is a marked increase in the level of calcitonin mRNA around E 18–19; all C cells exhibited intense reaction for calcitonin mRNA. After hatching, intensity of calcitonin mRNA expression was more and more increased. Northern blot analysis with the calcitonin probe also indicated that calcitonin synthesis of the C cells progressively increased with developmental gradient, and reached to the adult level at 1 month after hatching. On the other hand, intensity of hybridization signal of proenkephalin mRNA was maintained moderately during development. In contrast to enkephalin immunoreactivity, which is markedly decreased after hatching, proenkephalin mRNA expression was consistently detected in many C cells of 1- and 2-month-old chickens. Reverse transcription–polymerase chain reaction (RT–PCR) analysis confirmed that proenkephalin mRNA was obtained in the ultimobranchial glands of not only embryos but also 1-day- and 1-month-old chickens. Furthermore, Northern blot analysis demonstrated that a single band for proenkephalin mRNA was obtained in the poly (A) +RNA isolated from the ultimobranchial gland of 1-day-old chicks. Thus, the present study evidences that proenkephalin mRNA is synthesized in almost all C cells of chicken ultimobranchial glands throughout life. Enkephalin may be essential for C cell function.

Dopamine–opiate interaction in the regulation of neostriatal and pallidal neuronal activity as assessed by opioid precursor peptides and glutamate decarboxylase messenger RNA expression

Neostriatal GABAergic neurons projecting to the globus pallidus synthesize the opioid peptide enkephalin, while those innervating the substantia nigra pars reticulata and the entopeduncular nucleus synthesize dynorphin. The differential control exerted by dopamine on the activity of these two efferent projections concerns also the biosynthesis of these opioid peptides. Using in situ hybridization histochemistry, we investigated the role of opioid co-transmission in the regulation of neostriatal and pallidal activity. The expression of the messenger RNAs encoding glutamate decarboxylase—the biosynthetic enzyme of GABA—and the precursor peptides of enkephalin (preproenkephalin) and dynorphin (preprodynorphin) were measured in rats after a sustained blockade of opioid receptors by naloxone (s.c. implanted osmotic minipump, eight days, 3 mg/kg per h), and/or a subchronic blockade of D 2 dopamine receptors by haloperidol (one week, 1.25 mg/kg s.c. twice a day). The density of μ opioid receptors in the neostriatum and globus pallidus was determined by autoradiography. Naloxone treatment resulted in a strong up-regulation of neostriatal and pallidal μ opioid receptors that was not affected by the concurrent administration of haloperidol. Haloperidol alone produced a moderate down-regulation of neostriatal and pallidal μ opioid receptors. Haloperidol strongly stimulated the expression of neostriatal preproenkephalin and preprodynorphin messenger RNAs. This effect was partially attenuated by naloxone, which alone produced moderate increases in preproenkephalin and preprodynorphin messenger RNA levels. In the neostriatum, naloxone did not affect either basal or haloperidol-stimulated glutamate decarboxylase messenger RNA expression. A strong reduction of glutamate decarboxylase messenger RNA expression was detected over pallidal neurons following either naloxone or haloperidol treatment, but concurrent administration of the two antagonists did not result in a further decrease. The amplitude of the variations of μ opioid receptor density and of preproenkephalin and preprodynorphin messenger RNA levels suggests that the regulation of neostriatal and pallidal μ opioid receptors is more susceptible to a direct opioid antagonism, while the biosynthesis of opioid peptides in the neostriatum is more dependent on the dopaminergic transmission. The down-regulation of μ opioid receptors following haloperidol represents probably an adaptive change to increased enkephalin biosynthesis and release. The haloperidol-induced increase in neostriatal preprodynorphin messenger RNA expression might result from an indirect, intermittent stimulation of neostriatal D 1 receptors. The haloperidol-induced decrease of pallidal glutamate decarboxylase messenger RNA expression suggests, in keeping with the current functional model of the basal ganglia, that the activation of the striatopallidal projection produced by the interruption of neostriatal dopaminergic transmission reduces the GABAergic output of the globus pallidus. The reduction of pallidal glutamate decarboxylase messenger RNA expression following opioid receptor blockade indicates an indirect, excitatory influence of enkephalin upon globus pallidus neurons and, consequently, a functional antagonism between the two neuroactive substances (GABA and enkephalin) of the striatopallidal projection in the control of globus pallidus output. Through this antagonism enkephalin could partly attenuate the GABA-mediated effects of a dopaminergic denervation on pallidal neuronal activity.

Growth factors effects on the expression of morphological and biochemical properties of avian embryonic sympathetic cells. Emphasis on NGF

Growth factors are known to be important agents in the differentiation and modulation of neuronal phenotypes. We have analyzed the effect of several growth factors on the modulation of morphological and biochemical properties of avian embryonic sympathetic neurons. The growth factors studied include: nerve growth factor (NGF), neurotrophin-3 (NT-3), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and transforming growth factor β-1 (TGF-β1). Morphological properties were analyzed by immunocytochemistry to neurofilament proteins and visualization of fibers after glyoxylic acid-induced fluorescence. Biochemical modulation was determined by radioimmunoanalysis for the peptides enkephalin (ENK), somatostatin (SS) and neuropeptide Y (NPY) and by HPLC-electrochemistry quantification of catecholamines. Similar to previous results using chromaffin cell cultures [R. Ramı́rez-Ordóñez, J.E. Garcı́a-Arrarás, Peptidergic, catecholaminergic and morphological properties of avian chromaffin cells are modulated distinctively by growth factors, Dev. Brain Res., 87 (1995) 160–171], we found a dissociation in the modulation of biochemical and morphological properties, however, the effect of specific factors differed between the chromaffin and sympathetic cultures. We have focused on NGF to analyze its effect on the sympathetic peptide phenotypes and its lack of an effect on the chromaffin cell peptide phenotypes. The results presented here, establish interesting differences between chromaffin cells and sympathetic neurons that are of importance to studies of cell lineage and differentiation.

Enkephalin regulates acute D 2 dopamine receptor antagonist-induced immediate-early gene expression in striatal neurons

Projection neurons of the striatum release opioid peptides in addition to GABA. Our previous studies showed that the opioid peptide dynorphin regulates that subtype of projection neurons which sends axons to the substantia nigra/entopeduncular nucleus, as indicated by an inhibitory action of dynorphin/agonists on D 1 dopamine receptor-mediated immediate-early gene induction in these neurons. The other subtype of striatal projection neurons projects to the globus pallidus and contains the opioid peptide enkephalin. Here, we investigated whether enkephalin regulates the function of striatopallidal neurons, by analysing opioid effects on immediate-early gene induction by D 2 dopamine receptor blockade that occurs in these neurons. Thus, the effects of systemic and intrastriatal administration of various opioid receptor agonists and antagonists on immediate-early gene expression (c- fos, zif 268) induced by the D 2 receptor antagonist eticlopride were examined with in situ hybridization histochemistry. Intrastriatal infusion of enkephalin (delta and mu), but not dynorphin (kappa), receptor agonists suppressed immediate-early gene induction by eticlopride in a dose-dependent manner. This suppression was blocked by the opioid receptor antagonist naloxone, confirming the involvement of opioid receptors. Repeated treatment with D 2 receptor antagonists produces increased enkephalin expression and diminished immediate-early gene inducibility in striatopallidal neurons, as well as behavioral effects that are attenuated compared to those of acute treatment (e.g., reduced akinesia). Naloxone reversed such behavioral recovery (i.e. reinstated akinesia), but did not significantly affect suppressed immediate-early gene induction. Our results indicate that enkephalin acts, via mu and delta receptors in the striatum, to inhibit acute effects of D 2 receptor blockade in striatopallidal neurons. Moreover, the present findings suggest that increased enkephalin expression after repeated D 2 receptor antagonist treatment is an adaptive response that counteracts functional consequences of D 2 receptor blockade, but is not involved in suppressed immediate-early gene induction. Together with our earlier findings of the role of dynorphin, these results indicate that opioid peptides in the striatum serve as negative feedback systems to regulate the striatal output pathways in which they are expressed.

The relationship between oral dyskinesias produced by long-term haloperidol treatment, the density of striatal preproenkephalin messenger RNA and enkephalin peptide, and the number of striatal neurons expressing preproenkephalin messenger RNA in rats

Neuroleptic-induced oral dyskinesias in rats, a putative analogue to human tardive dyskinesia, may be due to excitotoxic degeneration within the striatum. Haloperidol treatment for 34 weeks increased the optical density of preproenkephalin messenger RNA in individual striatal neurons and enkephalin peptide in the neuropil, regardless of the level of oral dyskinesia produced. However, using unbiased stereological methods, an increased number of striatal neurons expressing preproenkephalin messenger RNA was observed only in rats that did not develop pronounced oral dyskinesias during haloperidol treatment. Said in another manner, the haloperidol-treated animals that developed pronounced oral dyskinesias, failed to produce an increase in the number of neurons expressing preproenkephalin messenger RNA. These results indicate that the mechanism by which neuroleptics induce oral dyskinesias in rats, and perhaps tardive dyskinesia in humans, involves a functional disturbance or even damage to a subpopulation of enkephalinergic neurons in the striatum.

Opiate and N-methyl-D-aspartate receptors in form-deprivation myopia.

Pharmacological studies have implicated retinal opiate pathways in the visual regulation of ocular growth. However, the effects of opiate receptor subtype-specific compounds on form-deprivation myopia (FDM) are inconsistent (Seltner et al., 1997), and may be mediated by non-opiate receptors. The purpose of this study was to test whether opiate receptor-inactive (D-) enantiomers elicit the same FDM-suppressing effect as their opiate receptor-active (L-) counterparts. Since some opiates are thought to act at NMDA receptors, we also tested whether NMDA receptor agonists and antagonists influence ocular growth or FDM. We found that both L- and D- enantiomers of morphine-like compounds (dextrorphanol and levorphanol, and D- and L-naloxone) were equally effective in blocking FDM. The NMDA receptor antagonists dextromethorphan, MK801, and AP5 also suppressed FDM. A single toxic dose of NMDA, that destroys many subtypes of amacrine cells (including those that synthesize the opioid peptide enkephalin), induced myopia and ocular enlargement in ungoggled eyes, and eliminated the ability of form-deprivation to enhance ocular growth. The NR-1 subunit of the NMDA receptor was localized to a narrowly stratified, intense stratum at approximately 50% depth in the inner plexiform layer, diffusely throughout the proximal inner plexiform layer, and to many somata in the amacrine and ganglion cell layers. These observations suggest that most effects of opiate receptor ligands on FDM in the chick are mediated by non-opiate receptors, which are likely to include NMDA receptors. NMDA as an excitotoxin transiently enhances ocular growth, but thereafter disables retinal mechanisms that promote emmetropization and FDM. These observations are consistent with a prominent role for pathways utilizing NMDA receptors in FDM and ocular growth-control.

Role of dynorphin and enkephalin in the regulation of striatal output pathways and behavior.

Projection neurons in the striatum give rise to two output systems, the "direct" and "indirect" pathways, which antagonistically regulate basal ganglia output. While all striatal projection neurons utilize GABA as their principal neurotransmitter, they express different opioid peptide co-transmitters and also different dopamine receptor subtypes. Neurons of the direct pathway express the peptide dynorphin and the D1 dopamine receptor, whereas indirect pathway neurons express the peptide enkephalin and the D2 receptor. In the present review, we summarize our findings on the function of dynorphin and enkephalin in these striatal output pathways. In these studies, we used D1- or D2-receptor-mediated induction of immediate-early genes as a cellular response in direct or indirect projection neurons, respectively, to investigate the role of these opioid peptides. Our results suggest that the specific function of dynorphin and enkephalin is to dampen excessive activation of these neurons by dopamine and other neurotransmitters. Levels of these opioid peptides are elevated by repeated, excessive activation of these pathways, which appears to be an adaptive or compensatory response. Behavioral consequences of increased opioid peptide function in striatal output pathways may include behavioral sensitization (dynorphin) and recovery of motor function (enkephalin).

Aminophosphinic inhibitors as transition state analogues of enkephalin-degrading enzymes: a class of central analgesics.

Inhibition of aminopeptidase N and neutral endopeptidase-24.11, two zinc metallopeptidases involved in the inactivation of the opioid peptides enkephalins, produces potent physiological analgesic responses, without major side-effects, in all animal models of pain in which morphine is active. Dual inhibitors of both enzymes could fill the gap between opioid analgesics and antalgics. Until now, attempts to find a compound with high affinity both for neutral endopeptidase and aminopeptidase N have failed. We report here the design of dual competitive inhibitors of both enzymes with KI values in the nanomolar range. These have been obtained by selecting R1, R2, and R3 determinants in aminophosphinic-containing inhibitors: NH2---CH(R1)P(O)---(OH)CH2---CH(R2)CONH---CH(R3)COOH, for optimal recognition of the two enkephalin inactivating enzymes, whose active site peculiarities, determined by site-directed mutagenesis, have been taken into account. The best inhibitors were 10x more potent than described dual inhibitors in alleviating acute and inflammatory nociceptive stimuli in mice, thus providing a basis for the development of a family of analgesics devoid of opioid side effects.

Gradient high-performance liquid chromatographic analysis of enkephalin peptides, their metabolites, and enzyme inhibitors using combined ultraviolet and electrochemical detection: II. Application to ocular permeability studies in vitro

A gradient HPLC method with combined ultraviolet (UV) and electrochemical detection (ED) was used to study the ocular permeability of [ d-Ala 2]-methionine enkephalinamide (MEA) in vitro. Coulometric ED was selective for MEA and its tyrosine-containing metabolites with quantitation limits between 20 and 60 n M (1.0–3.0 pmol per 50-μl injection), whereas UV detection at 205 nm allowed the determination of several aromatic metabolites and enzyme inhibitors with quantitation limits between 40 and 500 n M (2.0–25.0 pmol). This method was capable of detecting permeability of MEA and metabolite formation in the cornea and conjunctiva in vitro. Furthermore, effects of aminopeptidase inhibitor bestatin and enkephalinase inhibitor SCH 39370 on permeation and metabolism of MEA could be determined.

Selective innervation of different target tissues in guinea-pig cranial exocrine glands by sub-populations of parasympathetic and sympathetic neurons

This study has used multiple-labelling immunohistochemistry and quantitative analysis to examine the projections of subpopulations of parasympathetic and sympathetic neurons to different vascular and secretory structures in five cranial exocrine glands of guinea-pigs. Multiple subpopulations of parasympathetic axons, identified by immunoreactivity (IR) for various combinations of peptides, innervated arteries, arterioles, ducts and acini in sublingual, submandibular, parotid, lacrimal and zygomatic glands, although axons were absent from ducts in the parotid gland. Most parasympathetic axons contained IR for vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), with or without enkephalin (Enk). The proportion of parasympathetic axons that contained Enk-IR varied greatly between target tissues and glands: Enk-IR was more common in axons supplying secretory ducts, acini and arterioles than in axons innervating more proximal arteries; Enk-IR was less common in axons supplying the lacrimal gland than axons supplying the submandibular, lacrimal and zygomatic glands. Sympathetic axons with IR for tyrosine hydroxylase (TH) innervated arterial vessels in all glands, but innervated secretory structures only in the salivary glands. Sympathetic axons supplying proximal arterial segments often contained NPY-IR and sometimes also contained IR for dynorphin. Dynorphin-IR was more common in axons in the parotid, lacrimal and zygomatic glands than in the sublingual and submandibular glands. In contrast, axons supplying arterioles, ducts and acini lacked peptide IR. These results indicate that neuronal pathways regulating proximal arteries in cranial exocrine glands are different from the neuronal pathways regulating arterioles and acini, and may be different from neurons projecting to proximal secretory ducts. Furthermore, the peptides enkephalin, NPY and dynorphin are likely to make variable contributions to autonomic neurotransmission in different arterial segments and in different cranial exocrine glands.

Micellar electrokinetic chromatographic study of the interaction between enkephalin peptide analogs and charged micelles.

The relative hydrophobicity/hydrophilicity of the pentapeptides leucine enkephalin (LE), methionine enkephalin (ME) and five analogs, differing in their uncharged side chain and/or chirality, was investigated by micellar electrokinetic capillary chromatography (MEKC) employing anionic and cationic surfactants. The effect of sodium dodecyl sulfate (SDS) concentration on peptide mobility was studied at pH 8.8, a value that is well above the peptide isoelectric point, to minimize electrostatic interaction with the anionic micelles. Similarly, the effect of cetyltrimethylammonium bromide (CTAB) cationic micelles on peptide migration was studied at pH 4.1. The migration order from MEKC experiments was compared to the peptide hydrophobicity calculated from reversed-phase HPLC-derived hydrophobicity coefficients. Although relative peptide hydrophobicity was, in general, positively correlated with effective electrophoretic mobility, a tryptophan-containing analog showed only weak interaction with micelles compared to the less hydrophobic peptides. The enkephalins studied were zwitterionic in character from pH 3 to 8, and their migration as a function of pH under MEKC conditions demonstrated that electrostatic forces were at least as important as hydrophobic interactions in pentapeptide-micelle complexation.

Opiate modulation of dynorphin conversion in primary cultures of rat cerebral cortex

Rat brain cortical cells in primary culture were used to investigate long-term effects of opiates on endopeptidases acting on dynorphin peptides. Enzyme activity in the soluble fraction of the cells converted dynorphin B to Leu-enkephalin-Arg 6 and to a lesser extent to Leu-enkephalin. Five day treatment with 10 μM morphine increased the conversion to Leu-enkephalin-Arg 6 by 370%. This effect was prevented by the presence of naloxone in the culture medium. The opiate-inducible activity was directed to the Arg-Arg bond in dynorphins with preference for dynorphin B> α-neoendorphin>>dynorphin A. The K m for the generation of Leu-enkephalin-Arg 6 from dynorphin B was 40 μM. Enzyme activity was inhibited by dynorphin fragments, in the following order of potency: dynorphin A(1–13)>A(2–13)>A(1–17)>A(2–17) and by SH-reagents, suggesting the presence of a cysteine-protease. The opiate-stimulated dynorphin-converting enzyme (DCE)-activity affects the balance between dynorphin peptides (selective for κ-opioid receptors) and enkephalin peptides (selective for δ-opioid receptors). Since both types of opioid peptides can influence the development of opiate tolerance, the change in the extent of this transformation may be functionally important.

Long-term potentiation in perforant path/granule cell synapses is associated with a post-synaptic induction of proenkephalin gene expression

Enkephalin peptides released from hippocampal mossy fibres lower the threshold for the generation of long-term potentiation (LTP) at the mossy fibre synapses. High frequency stimulation of the hippocampal dentate gyrus, sufficient to induce mossy fibre LTP, is associated with increased expression of the proenkephalin gene in the granule cells. We show here that a similar elevation in proenkephalin mRNA levels is observed, in anaesthetised rats, following stimulation of the perforant path sufficient to induce LTP in the perforant path/granule cell synapses. This strengthens the evidence implicating granule cell enkephalins as mediators of functional plasticity in the hippocampus. Furthermore, the results hint at a form of `domino plasticity', where potentiation of transmission at the perforant path/granule cell synapses is subsequently followed by an enkephalin-mediated potentiation of transmission at the mossy fibre synapses.

In Situ and Immunohistochemical Analysis of Endogenous Opioid Receptor mRNA and Protein Expression in the Developing Human Brain. • 1745

In the extremely premature neonate, much CNS development is undertaken ex utero, while the infant is exposed to multiple environmental and pharmacological stressors. Although there is a wealth of information concerning long-term outcomes of premature survivors, little is known regarding the effects of environmental influences on their developing brain, particularly on their developing opioid receptor systems. Premature neonates are regularly exposed to exogenous opiates (e.g., morphine, fentanyl) while critically ill, and this may permanently alter their opioid receptor profile as adults. In order to better study this, it is important to first learn the expression of opioid receptors during normal human CNS development. Fresh post-mortem brain specimens were obtained at various gestational ages (16 to 36 wk). Brain specimens were blocked, immersed in Zamboni's fixative for 100-200 days, soaked in 10% sucrose, quick-frozen in isopentane, then cryostat cut into 15μm sections. For human mu immunohistochemistry, tissue was incubated in primary rabbit antiserum directed against the C-terminal sequence of the rat mu receptor protein, then it was visualized using nickel chloride and diaminobenzidine. Pre-absorption controls were performed using 2μM protein absorption of primary antiserum prior to tissue incubation. For in situ hybridization, (35)S-labeled cRNA probes were generated against the human mu and kappa receptors and enkephalin peptide. After in situ hybridization was completed, sections were then opposed to X-ray film for several weeks prior to developing. Labeling of mRNA in parietal cortex was robust for enkephalin peptide in tissue at all gestational ages, and at 20-36 weeks for kappa receptor. Mu receptor mRNA was not detected in this area until 25 weeks gestation. Striatal labeling was strong at 16 weeks using enkephalin probe, but no kappa or mu mRNA was seen in this area until after 22 weeks. Mu immunostaining was observed in the striatum, entorhinal and cerebral cortex, and several additional forebrain and brainstem nuclei These regions are similar to those described previously in rat. Comparisons to distribution of mu receptor in the developing rat brain is also discussed. Supported by NIDA Grant T32DA07268.

The projection from the striatum to the nucleus basalis in the rat: an electron microscopic study

Previous studies have shown that the striatum provides synaptic inputs to the globus pallidus and entopeduncular nucleus in which GABA is co-localized with the peptides enkephalin and substance P. The aim of this study in the rat was to determine whether the striatal projections also make synaptic contact with the cholinergic neurons of the nucleus basalis, which lie near to the pallidal areas in the rat brain. The anterograde tracer biocytin was injected into different parts of the striatum, and brain sections were stained for biocytin and choline acetyltransferase immunoreactivity by using a dual colour method. Terminals labelled with biocytin by anterograde transport and which made synaptic contact with choline acetyltransferase-positive soma and dendrites were identified by light–electron microscopic correlation methods. In the cases where the biocytin injections had been made in the dorsal or lateral striatum, biocytin-labelled terminals made synaptic contact with cholinergic cells in the region between the main termination zones in the globus pallidus and the entopeduncular nucleus. In the cases where the injections had been made in the ventromedial and posterior striatum, there was greater overlap between choline acetyltransferase-positive structures and biocytin-labelled terminals in the main termination zones in the globus pallidus or entopeduncular nucleus, but relatively few of these terminals made synaptic contacts on to the cholinergic neurons. The results therefore indicate that the cholinergic nucleus basalis cells receive a relatively sparse synaptic input from all parts of the striatum. It has recently been shown that the cholinergic cells of the nucleus basalis selectively express high levels of substance P and opioid receptor messenger RNAs, while the non-cholinergic pallidal cells have much higher levels of GABA A receptor subunit messenger RNAs. It is concluded that the cholinergic neurons of the nucleus basalis in the rat may be selectively responsive to the peptidergic components of the striatal outputs, and that they are most likely to be influenced by both the limbic and sensorimotor parts of the striatum.

Gradient high-performance liquid chromatographic analysis of enkephalin peptides, their metabolites and enzyme inhibitors using combined ultraviolet and electrochemical detection.: I. Optimization of separation and detection

Coulometric electrochemical detection (ED) was compatible with gradient HPLC analysis of enkephalin peptides, their metabolites and several enzyme inhibitors with combined UV detection. The compounds were separated using a reversed-phase octylsilane column with 35 m M phosphate buffer (pH 2.1)–acetonitrile eluent. Hydrodynamic voltammograms were determined for [ d-Ala 2]–methionine enkephalinamide (MEA) and its metabolites, and the response of MEA consisted of the oxidation of the tyrosine and methioninamide residues with a total transfer of five electrons at the highest potential used. Based on the voltammograms, ED was optimized for the selective determination of MEA and other tyrosine-containing peptides with detection limits between 0.4 and 0.6 pmol per 50 μl injection. In addition, simultaneous UV detection at 205 nm allowed the determination of all the aromatic peptides and enzyme inhibitors with detection limits between 1.5 and 30.0 pmol.

Enkephalins in hematopoiesis

Recent data support the view that neuropeptide mediators, in particular opioid peptides, participate in the control of hematopoiesis. The main arguments are: neuropeptides modulate the functions of lymphoid cells, macrophages and mature granulocytes; they control cell proliferation and differentiation in many tissues, particularly during embryogenesis; lymphoid cells, macrophages, polymorphonuclear granulocytes and bone marrow stromal elements express neuropeptide receptors; bone marrow cells produce opioid-like neuropeptides; the CD10/CALLA marker of lymphoid, myeloid and marrow stromal cells is an enzyme, endopeptidase, which cleaves- and thus activates/inactivates-opioid and other neuropeptides. We have shown that opioid peptides enkephalins, opioid antagonist naloxone, and the inhibitor of enkephalin-degrading endopeptidase, thiorphan, modulate the proliferation and differentiation of hematopoietic cells in clonal and long-term cultures of mouse bone marrow. The effects partly depended on the presence of the accessory hematopoietic elements, and followed a circadian pattern. The dose-responses were irregular, showed strain-dependent and individual variations, and apparently reflected the state of the activity of target cells, cellular interactions and simultaneous signals by other mediators. The enkephalins were shown to bind to specific (opioid) receptors on the target cells, and their signals to be transmitted to the cell interior by a cascade of secondary messengers including diacyl-glycerol (DAG), protein-kinase C (PKC) and Ca++ ions. Neuropeptide regulation of hematopoiesis might belong to a complex immuno-neuroendocrine network including melatonin.

Carboxypeptidase E Activity Is Deficient in Mice with the fat Mutation: EFFECT ON PEPTIDE PROCESSING

Carboxypeptidase E (CPE) is involved in the biosynthesis of many peptide hormones and neurotransmitters. Mice with the fat mutation have previously been found to have a point mutation in the cpe gene, and to have greatly reduced levels of CPE-like enzyme activity in the pituitary and pancreatic islets (Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). In the present report, we examined CPE-like activity and peptide processing in several tissues of C57BLKS/LtJ-Cpefat/Cpefat mutant (Cpefat/Cpefat) mice. Whereas CPE-like activity is detected in homogenates of Cpefat/Cpefat mouse tissues, the majority of this activity is not due to CPE based on the sensitivity to p-chloromercuriphenyl sulfonate. In addition, the Cpefat/Cpefat activity does not bind to a substrate affinity column under conditions that bind CPE. Furthermore, the enzyme activity and immunoreactive properties of the activity purified from Cpefat/Cpefat brain are distinct from those of CPE. Taken together, these data suggest that CPE is completely inactive in the Cpefat/Cpefat mice, and that all of the CPE-like activity is due to other carboxypeptidases such as carboxypeptidase D. Levels of Leu-enkephalin in Cpefat/Cpefat mouse brain are approximately 5-fold lower than those in control brain. Treatment of the Cpefat/Cpefat brain extract with carboxypeptidase B restores the level of Leu-enkephalin to the level in control brain. Interestingly, the large molecular weight enkephalin-containing peptides are elevated 2-3-fold in Cpefat/Cpefat mouse brain. These data indicate that CPE plays an important role in the processing of peptide hormones in various tissues, but that other carboxypeptidases also contribute to peptide processing. Furthermore, the increase in levels of high molecular weight enkephalin peptides in the Cpefat/Cpefat mouse suggests that CPE is required for efficient peptide processing by the endopeptidases.

Multivalent ligand system carrying enkephalin and neurotensin coimmobilized on liposomes.

A multivalent ligand system was constructed by coimmobilization of two kinds of peptide ligands, enkephalin and neurotensin derivatives having a dioctadecyl group, on dimyristoylphosphatidylcholine (DMPC) liposomes. The enkephalin derivatives are Tyr-D-Ala-Gly-Trp-Leu-(Sar-Sar-Pro)n-[N(C18H37)2] (Enk3nD, n = 0, 1, 2), where a dioctadecyl group was connected to the C-terminal side of enkephalin directly or through a hydrophilic and flexible spacer chain of different lengths. The neurotensin derivatives are Ac-Glu[N(C18H37)2l-(Sar-Sar-Pro)n-Arg-Arg-Pro-Tyr-Ile-Leu-OH (D3nNT, n = 0, 1, 2, 3). The derivatives were spontaneously immobilized on DMPC liposomes by overnight incubation. The receptor affinity of the enkephalin derivatives became significantly higher upon immobilization on liposomes. The highest affinity was obtained for the delta receptor by Enk6D immobilized on DMPC liposomes. This affinity is higher than that of enkephalinamide. Neurotensin derivatives coimmobilized with large amounts of Enk3D on the DMPC liposomes show higher affinity than the neurotensin derivatives immobilized alone. The effect of Enk3D on the receptor affinity of the coimmobilized neurotensin derivative disappeared by the addition of [Ala2, MePhe4, Glyol5]enkephalin (DAGO). Therefore, the receptor affinity of a peptide hormone is altered by immobilization on DMPC liposomes and by coimmobilization with other peptide hormones. It was confirmed by fluorescent microscopy that the multivalent ligand system binds to receptors without release of the bound ligands from DMPC liposomes.