Oxytocin in the periaqueductal gray participates in pain modulation in the rat by influencing endogenous opiate peptides.

Periaqueductal gray (PAG) plays a very important role in pain modulation through endogenous opiate peptides including leucine-enkephalin (L-Ek), methionine-enkephalin (M-Ek), ß-endorphin (ß-Ep) and dynorphin A(1-13) (DynA(1-13)). Our pervious study has demonstrated that intra-PAG injection of oxytocin (OXT) increases the pain threshold, and local administration of OXT receptor antagonist decreases the pain threshold, in which the antinociceptive role of OXT can be reversed by pre-PAG administration of OXT receptor antagonist. The experiment was designed to investigate the effect of OXT on endogenous opiate peptides in the rat PAG during the pain process. The results showed that (1) the concentrations of OXT, L-Ek, M-Ek and ß-Ep, not DynA(1-13) in the PAG perfusion liquid were increased after the pain stimulation; (2) the concentrations of L-Ek, M-Ek and ß-Ep, not DynA(1-13) in the PAG perfusion liquid were decreased by the OXT receptor antagonist; (3) the increased pain threshold induced by the OXT was attenuated by naloxone, an opiate receptor antagonist; and (4) the concentrations of L-Ek, M-Ek and ß-Ep, not DynA(1-13) in the PAG perfusion liquid were increased by exogenous OXT administration. The data suggested that OXT in the PAG could influence the L-Ek, M-Ek and ß-Ep rather than DynA(1-13) to participate in pain modulation, i.e. OXT in the PAG participate in pain modulation by influencing the L-Ek, M-Ek and ß-Ep rather than DynA(1-13).

Rapid magnetic catch-and-release purification by hydrophobic interactions.

A reversible, conventional, and rapid purification method of hydrophobically tagged products using hydrophobic magnetic nanoparticles was developed. The reversible purification system entails simply controlling the polarity of solvents. First, for the catching procedure, poor solvents were added into a well-dispersed system of magnetic nanoparticles and tagged products. Once the poor solvents were added to the system, the products were recrystallized among the nanoparticles and the aggregation of magnetic nanoparticles occurred due to hydrophobic interactions. These aggregates with the products contained within them were able to be collected rapidly by magnets. Then, the releasing procedure can be easily performed by redispersing the collected aggregates into good solvents. The availability of this purification protocol was confirmed by using a hydrophobically tagged fluorescent model product. Furthermore, this rapid purification method was successfully applied to a peptide elongation reaction system which enabled the synthesis of peptides such as Leu-Enkephalin in high purity, in high yield, and in a short time.

Temporal changes of preproenkephalin mRNA and leu-enkephalin-like immunoreactivity in the neurons of the caudal spinal trigeminal nucleus and upper cervical cord after noxious stimuli.

The changing levels of preproenkephalin (PPE) mRNA expression and leu-enkephalin-like immunoreactivity (L-ENK-LI) in the caudal spinal trigeminal nucleus (Vc) and the upper cervical cord subsequent to subcutaneous injection of formalin into orofacial region were examined using in situ hybridization histochemistry and immunohistochemistry. Formalin injection resulted in a significant increase in the number of neurons expressing PPE mRNA in the superficial (laminae I-II) and deep (lamina V) layers of the ipsilateral Vc and the first cervical cord (C1). The expression of PPE mRNA increased 2 h and 4 h after formalin injection in the neurons of deep layer and superficial layers of the Vc, respectively. An increased expression of PPE gene could still be observed 48 h after formalin injection. The PPE mRNA expression in the neurons of the C1 had similar temporal changes as in the Vc. In parallel, the L-ENK-LI did not show any significant changes in the Vc and C1. These results indicate that peripheral noxious stimulation trans-synapticaly activates the expression of PPE gene in the neurons of the Vc and C1 and suggest that enkephallin (ENK) plays an important role in the processing and modulation of the oroficial noxious stimulation.

State-dependent changes of brain endogenous opioids in mammalian hibernation.

Endogenous opioids belonging to three opioid families were measured in different states of the hibernation cycle in brain of the Columbian ground squirrels. Using high-performance liquid chromatography-EC detection, the hypothalamic and septal concentrations of met-enkephalin were found to be significantly higher (p < 0.05) in the hibernating state than that in the nonhibernating state. In contrast, met-enkephalin content in the medulla decreased significantly during hibernation. Leuenkephalin content was only increased in the hypothalamus of hibernating animals. Using radioimmunoassay, dynorphin A-like immunoreactivity was observed to increase in the claustrum and striatum, whereas beta-endorphin-like peptides showed a significant increase in the hypothalamus during hibernation. It is evident that the changes in endogenous opioids in brain during hibernation are state dependent, type specific and region specific. These specific alterations of various endogenous opioids may imply their different roles in hibernation.

[Leu5]enkephalin-encoding sequences are targets for a specific DNA-binding factor.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.

The effect of prolonged treatment with imipramine and electroconvulsive shock on the levels of endogenous enkephalins in the nucleus accumbens and the ventral tegmentum of the rat.

The present study was designed to find out whether the prolonged administration of imipramine (IMI) or electroconvulsive shock (ECS) influences levels of endogenous enkephalins in the nucleus accumbens (NAS) and the ventral tegmentum (VTA) of the rat. Ressults indicate that treatment with IMI as well as with ECS has a profound effect on the levels of enkephalins in both structures. In the NAS both treatments lead to an increase in the levels of endogenous enkephalins and this effect is accompanied by an increase in mRNA coding for proenkephalin (measured by in situ hybridization) in this structure, indicating the enhancement of biosynthesis of endogenous enkephalinergic peptides following antidepressant treatment. The results are discussed in the light of the hypothesis concerning the influence of endogenous enkephalins on mesolimbic dopamine neurons, the activity of which plays a crucial role in the etiology of depression.

Aging, cardiac proenkephalin mRNA and enkephalin peptides in the Fisher 344 rat.

Cardiac proenkephalin (PENK) mRNA, methionine-enkephalin (ME) and leucine-enkephalin (LE) were determined from 2 days of age through senescence in Fisher 344 rats. Tissues were collected at 2 days, 2 weeks, 1, 2, 3, 7, 19, and either 22 or 27 months of age. Hearts were dissected, extracted and assayed for ME and LE by radioimmunoassay (RIA) or for PENK mRNA by Northern blot analysis with a cDNA probe. Relative left ventricular (LV) PENK mRNA was low in 2 day animals and increased slowly between 2 weeks and 3 months of age. LV PENK mRNA then rose five to six-fold between 3 and 27 months of age. LV ME measurements were high in neonatal animals, declined to a nadir during development and then rose again as the animals matured and advanced in age. The pattern for right ventricular (RV) ME was similar. Atrial ME, also high at 2 days, declined thereafter and remained low. LE measurements in LV, RV and the atria followed patterns similar to those described for ME. To evaluate for peptides contributed by cardiac nerves, 3, 7 and 22-month-old animals were acutely sympathectomized for 24 h with 6-hydroxydopamine. No decline in LV ME and LE was observed in the 6-hydroxydopamine treated animals. These data suggest several conclusions regarding myocardial enkephalinergic systems: (a) tissue enkephalin and PENK mRNA increase with advancing age, (b) tissue enkephalins may not strictly correlate with the relative abundance of PENK mRNA, and (c) most myocardial enkephalins are non-adrenergic in origin. The age-associated patterns in both PENK mRNA, ME and LE suggest that physiological, maturational or behavioral events between 3 and 7 months of age initiate the up-regulation and subsequent expansion of cardiac enkephalinergic systems.

Detection of Met-enkephalin in the pars intermedia of the lampreys, Ichthyomyzon castaneus and Petromyzon marinus.

Acid extracts of the brain and pars intermedia of the chestnut lamprey, Ichthyomyzon castaneus, were fractionated by a combination of gel filtration chromatography and reversed-phase HPLC, and screened with RIAs specific for Met-enkephalin and Leu-enkephalin, respectively. In the brain extract, both Met-enkephalin and Leu-enkephalin were detected in a molar ratio of approximately 4:1. These results would suggest that these two enkephalins were derived from a lamprey Proenkephalin precursor. However, reversed-phase HPLC analysis of the pars intermedia of this species revealed the presence of Met-enkephalin, but not Leu-enkephalin. Analysis of the pars intermedia of the marine lamprey, Petromyzon marinus, also indicated the presence of Met-enkephalin but not Leu-enkephalin. These results would suggest that the Met-enkephalin present in the pars intermedia of lampreys may not be derived from Proenkephalin, but may originate from another opioid precursor. This possibility and alternative hypotheses to explain these observations are discussed.

Occurrence of immunoreactive Met- and Leu-enkephalin-like peptides in the ovary of the green frog, Rana esculenta.

In the present study, we have localized for the first time Met- and Leu-enkephalin-like material in the ovary of the anuran, Rana esculenta, using the indirect immunofluorescence method. The ovaries were sampled during the main representative phases of the annual reproductive cycle of the frog, living in a mountain pond (Colfiorito, Umbria at 820 m a.s.l.). Strong immunoreactivity to Met- and Leu-enkephalin antisera was observed in the follicle cells of the granulosa layer of vitellogenic oocytes; moreover, during this phase, immunofluorescent materials were also radially localized in the cytoplasm and in the perinuclear zone. The mature oocytes showed Met- and Leu-enkephalin-like immunostaining in the thecal layer and in several granules scattered in the peripheral zone of the yolk. The different pattern of Leu- and Met-enkephalin-like immunoreactivity in the frog ovary parallels and complements the changes occurring in the reproductive (May) and in the vitellogenetic (September) phases during the ovarian cycle. Consequently, these findings strongly support the hypothesis for a local synthesis of these peptides in the ovary and suggest their possible involvement in the control of ovarian function.

Penicillin acylase-catalyzed protection and deprotection of amino groups as a promising approach in enzymatic peptide synthesis.

Penicillin acylase from E. coli is able to catalyze both the introduction and the removal of the phenylacetyl group. We have established that phenylacetyl derivatives of amino acids and peptides can be used in protease-catalyzed peptide synthesis. Here the synthesis of leucine-enkephalin using enzymes for N-terminal amino group protection, peptide bond formation and deprotection is described.

Studies of specificity and inhibition of human cerebrospinal fluid dynorphin converting enzyme.

Dynorphin-converting activity was recently discovered in human cerebrospinal fluid. This enzyme (hCSF-DCE) cleaves dynorphin A, dynorphin B and alpha-neoendorphin to release Leu-enkephalin-Arg6. To characterize the enzyme further we used several protease inhibitors, including N-peptidyl-O-acyl hydroxylamines which are known to act as potent irreversible inhibitors of serine and cysteine proteinases. No irreversible inactivation occurred but strong, reversible effects on the dynorphin-converting activity by some of the inhibitors tested could be observed. Although, hCSF-DCE binds its substrates (dynorphin A and B) in the microM-mM concentration range, it exhibits high specificity in recognizing and cleaving the linkage between the two basic amino acids in the substrate sequence.

Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector.

To examine the validity of the strategy to express a foreign gene as a fusion protein with the coat protein (CP) of tobacco mosaic virus (TMV), we have constructed ENK RNA by using an in vitro transcription system of TMV RNA. ENK RNA differs from TMV RNA only in that ENK RNA carries an additional sequence coding for Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) (Enk) with a preceding in-frame methionine just before the termination codon of CP gene. In protoplasts inoculated with ENK RNA, CP + Enk fusion protein accumulated as the major protein.

Formation of [Leu5]enkephalin from dynorphin A(1-8) by rat central nervous tissue in vitro.

[3H]Dynorphin A(1-8) is readily metabolised by rat lumbosacral spinal cord tissue in vitro, affording a variety of products including a significant amount (20% recovered activity) of [3H][Leu5]enkephalin. In the presence of the peptidase inhibitors bestatin, captopril, thiorphan, and leucyl-leucine, [3H][Leu5]enkephalin was the major metabolic product, accounting for 60% of recovered activity. Production of [3H][Leu5]enkephalin was seen across all gross brain regions. The enzyme responsible for the cleavage has an optimal substrate length of 8-13 amino acids and is inhibited by N-[1-(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a site-directed inhibitor of the metalloendopeptidase EC 3.4.24.15. However the enzymic breakdown also has properties in common with involvement of endo-oligopeptidase A. Possible consequences of the formation of [Leu5]-enkephalin from the smaller dynorphins are discussed.

Conversion of the dynorphins to Leu-enkephalin in human spinal cord.

This study describes three different endopeptidases in human spinal cord with high affinity for the dynorphins. The enzymes, with strict specificity towards paired basic residues, release Leu-enkephalin and its C-terminal extensions - Arg6 and -Arg6 -Arg7 from the substrate peptides. They differ with regard to molecular size, charge properties, and inhibitory profile. Two of the enzymes show optimal activity at neutral pH, whereas the third enzyme has its activity optimum at pH 5.6. They are all suggested to be of importance for the formation of Leu-enkephalin from prodynorphin.

Change of Leu-enkephalin- and B-endorphin-like immunoreactivity in the hippocampus after electroconvulsive shock and electroacupuncture.

To determine whether the effect of electroacupuncture (EA) on electroconvulsive shock (ECS) was through affecting endogenous opioid peptidergic system in the hippocampus, we used radioimmunoassay (RIA) to measure leu-enkephalin- and beta-endorphin-like immunoreactivity (L-E-LI and Beta-E-LI) in the hippocampal perfusate after ECS and EA treatments. Wistar rats were given ECS stimulation, which caused behavioral and electroencephalogram (EEG) convulsions. However, EA could suppress ECS-elicited convulsions (including both behavioral and EEG abnormalities). The results of RIA showed that after repeated ECS treatments, the contents of L-E-LI and Beta-E-LI increased significantly by 136% and 157%, respectively. In contrast, EA treatment depleted alterations of L-E-LI and Beta-E-LI induced by ECS, depleting L-E-LI by 32% and Beta-E-LI by 85%. These observations suggest that anticonvulsive action of EA is related to the release of L-E and Beta-E in hippocampus, probably through decreasing their release, thus to exert EA action.

The expression of gamma-aminobutyric acid and Leu-enkephalin immunoreactivity in primary monolayer cultures of rat striatum.

Primary monolayer cultures of rat striatum were examined for gamma-aminobutyric acid (GABA) and leucine-enkephalin (L-ENK) immunoreactivity. Cultures were established on polycation-treated glass coverslips from the striata of gestational day 17 rat embryos using a serum and insulin-supplemented medium. The proportion of GABA-immunoreactive (GABA-IR) neurons increased during the first week in vitro from approximately one third to nearly one half and remained relatively constant thereafter. On the other hand, the proportion of L-ENK-IR neurons increased gradually over the culturing period, increasing from about one-fifth of the neurons initially to one-half after 3-4 weeks in vitro. The changes in the proportions of GABA- and L-ENK-IR neurons appeared to be largely a consequence of the death of non-immunoreactive neurons, not delayed expression or induction of GABA or L-ENK traits. Light microscopic analysis of somatic-proximal neuritic morphology led to a partitioning of the neuronal population into 4 groups. GABA- and L-ENK-IR groups were heterogeneous in this regard and differed only modestly.

Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leuenkephalin.

In order to develop the use of carboxypeptidase Y (CPD-Y, EC 3.4.12.1) as a catalyst for radioactive hormonal synthesis, the stepwise synthesis of a pentapeptide, Leu-enkephalin, was studied on a microscale. Each peptide bond was formed by enzymatic catalysis, using microquantities of the precursors (amino acid or peptide esters as acyl-components and amino acid ester or amide as nucleophiles). The high condensation yields obtained suggests that CPD-Y can be a useful tool for preparation of radioactive hormonal peptides.

Development of transsynaptic regulation of adrenal enkephalin.

Transsynaptic activity differentially regulates biosynthesis of sympathoadrenal catecholamines and co-localized opiate peptides in the rat. We determined whether similar mechanisms were operative during development. Adrenal Leu-enkephalin (LEU), was first detected at E16.5, then increased 5-fold during maturation from birth to adulthood while adrenal weight increased 10-fold. Since medullary cells do not divide after the first postnatal week, this represents a specific maturational increase in LEU content per chromaffin cell. In adult medullae, decreasing transsynaptic activity through adrenal denervation or explantation results in a 30-50-fold increase in LEU. In contrast, LEU levels in denervated or explanted medullae from neonatal rats (less than or equal to 10 days) do not. Prolonged denervation (day 5-21) prevented even the normal maturational increase in LEU. However, depolarizing medullae with KCl lowered LEU levels at all ages tested with an increased magnitude of effect after 10 days postnatal age. Specific deficits in signal-transduction mechanisms or immaturity of opiate biosynthetic pathways may account for these observations. Thus, during development, adrenal opiate peptides are not under transsynaptic control yet require presynaptic terminals to mature normally. Therefore, like catecholamines, co-localized adrenal opiate peptides require presynaptic regulatory signals to achieve normal development and function.