Cellular localization of proenkephalin mRNA and enkephalin peptide products in cultured astrocytes

To identify the possible cellular sites of opioid gene expression during ontogeny, proenkephalin mRNA and enkephalin peptide expression were examined, respectively, by in situ hybridization and immunocytochemistry in organotypic explants of rat cerebellum and in astrocyte-enriched cultures of murine cerebral hemispheres. High levels of proenkephalin mRNA and enkephalin immunoreactivity were detected in immature cells identified as astrocytes. Double-labeling studies combining in situ hybridization and immunocytochemical localization of the astrocytic marker, glial fibrillary acidic protein, provided direct evidence that proenkephalin mRNA is expressed by astrocytes in culture. Based on previous studies that Met-enkephalin can inhibit astrocyte growth in vitro, the present results suggest that proenkephalin gene expression by astrocytes is important during central nervous system maturation.     

Immunocytochemical localization of enkephalins in the brain of the African lungfish,Protopterus annectens,provides evidence for differential distribution of Met-enkephalin and Leu-enkephalin

The distribution of various opioid peptides derived from proenkephalin A and B was studied in the brain of the African lungfish Protopterus annectens by using a series of antibodies directed against mammalian opioid peptides. The results show that both Met-enkephalin- and Leu-enkephalin-immunoreactive peptides are present in the lungfish brain. In contrast, enkephalin forms similar to Met-enkephalin-Arg-Phe, or Met-enkephalin-Arg-Gly-Leu, as well as mammalian α-neoendorphin, dynorphin A (1–8), dynorphin A (1–13), or dynorphin A (1–17) were not detected. In all major subdivisions of the brain, the overwhelming majority of Met-enkephalin- and Leu-enkephalin-immunoreactive cells were distinct. In particular, cell bodies reacting only with Leu-enkephalin antibodies were detected in the medial subpallium of the telencephalon, the griseum centrale, the reticular formation, the nucleus of the solitary tract, and the visceral sensory area of the rhombencephalon. Cell bodies reacting only with Met-enkephalin antibodies were found in the lateral subpallium of the telencephalon, the caudal hypothalamus, and the tegmentum of the mesencephalon. The preoptic periventricular nucleus of the hypothalamus exhibited a high density of Met-enkephalin-immunoreactive neurons and only a few Leu-enkephalin-immunoreactive neurons. The distribution of Met-enkephalin- and Leu-enkephalin-immunoreactive cell bodies and fibers in the lungfish brain showed similarities to the distribution of proenkephalin A-derived peptides described previously in the brain of land vertebrates. The presence of Met-enkephalin- and Leu-enkephalin-like peptides in distinct regions, together with the absence of dynorphin-related peptides, suggests that, in the lungfish, Met-enkephalin and Leu-enkephalin may originate from distinct precursors. J. Comp. Neurol. 396:275–287, 1998. © 1998 Wiley-Liss, Inc.     

Haloperidol increases proenkephalin mRNA levels in the caudate-putamen of the rat: a quantitative study at the cellular level using in situ hybridization

Previous immunocytochemical studies have shown that the opioid peptides, Met-enkephalin and Leu-enkephalin, are present in medium-sized, spiny projection neurons of the caudate-putamen. It has also been demonstrated that chronic treatment of rats with the dopamine receptor blocker, haloperidol, results in an increase in the levels of enkephalin peptides and proenkephalin mRNA in this brain region. To determine whether this increase in proenkephalin mRNA content is exhibited by all enkephalinergic neurons of the caudate-putamen or by only a subpopulation, we have used in situ nucleic acid hybridization to examine the haloperidol-induced increase in proenkephalin mRNA levels at the cellular level. Results of in situ hybridization suggest that all enkephalinergic neurons in the caudate-putamen can respond to haloperidol treatment with an increase in steady state levels of proenkephalin mRNA, and that the mean induction is an approximate 3-fold increase in the message levels. This suggests that dopamine exerts a tonic inhibitory effect on the expression of the proenkephalin gene in all of the enkephalinergic neurons of the caudate-putamen. Dot blot analysis indicated a 2.4-fold increase in the tissue levels of this mRNA. The agreement between the in situ hybridization results and dot blot analysis supports in situ hybridization as a reliable method for quantitative studies of alterations in neuropeptide precursor mRNAs in the brain.     

Basal release of Met-enkephalin and neurotensin in the ventrolateral periaqueductal gray matter of the rat: a microdialysis study of antinociceptive circuits

The periaqueductal gray (PAG) contains neural circuits that participate in descending antinociception. Anatomical and electrophysiological evidence suggests that these circuits might employ opioid peptides and GABA in series to remove a tonic inhibition of descending PAG output neurons. The present studies examined the release of the antinociceptive peptides Met-enkephalin and neurotensin in the ventrolateral PAG, and investigated the interaction between GABA and Met-enkephalin release. In awake and freely moving rats the ventrolateral PAG was dialysed using 25 ga. concentric probes. Basal release of peptide in 12 min or 40 min fractions was determined using radioimmunoassays. To establish how the ventrolateral PAG responds to nociception, dialysis was performed following unilateral hindpaw inflammation using Complete Freund's Adjuvant. Twenty-four hours after inflammation was induced, neurotensin release was increased 133% and Met-enkephalin release was increased 353% compared to control animals. Seven days after inflammation was induced, neurotensin release declined precipitously, while basal Met-enkephalin release remained elevated 313% above controls. Thus, unlike enkephalin, increased basal neurotensin release is not sustained with persistent tonic nociception. In addition, we confirmed in normal animals that the ventrolateral PAG is induced to release Met-enkephalin by systemic morphine. A 43% increase in basal Met-enkephalin release was observed immediately following a 12 mg/kg i.p. morphine injection. Morphine should have the opposite effect (inhibit peptide release) if it acts directly on the enkephalinergic neurons. Thus, we examined the hypothesis that GABAergic interneurons in the PAG mediated morphine-stimulated enkephalin release. When the GABAantagonist bicuculline (0.25 μM to 25 μM) was co-infused with the dialysis medium, Met-enkephalin release increased in a dose-dependent fashion and peaked 68% above pre-infusion levels. These data elucidate the reciprocal inhibitory relationship between GABA and enkephalin in the ventrolateral PAG. We hypothesize that, when nociception induces Met-enkephalin release within this region, the tonic GABAergic inhibition is overcome, resulting in greater sensitivity of PAG enkephalinergic neurons. Ultimately, this enhanced enkephalin release should result in greater excitability of the descending PAG output neurons that are responsible for antinociception.     

Deciphering the origin of Met-enkephalin and Leu-enkephalin in Lobe-finned fish: cloning of australian lungfish proenkephalin

The previous detection of Met-enkephalin and Leu-enkephalin in the CNS of the Australian lungfish, Neoceratodus forsteri, in a molar ratio comparable to mammals suggested that the lungfish proenkephalin precursor should contain the sequences of both Met-enkephalin and Leu-enkephalin as seen for mammalian proenkephalin. However, the cloning of a full-length proenkephalin cDNA from the CNS of the Australian lungfish indicates that the organization of this precursor is more similar to amphibian proenkephalin than mammalian proenkephalin. The Australian lungfish cDNA is 1284 nucleotides in length and the open reading frame (267 amino acids) contains seven opioid sequences (GenBank #AF232671). There are five copies of the Met-enkephalin sequence flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended forms of Met-enkephalin: YGGFMRSL and YGGFMGY. As seen for amphibians, no Leu-enkephalin sequence was detected in the Australian lungfish proenkephalin cDNA. The fact that Leu-enkephalin has been identified by radioimmunoassay and HPLC analysis in the CNS of the Australian lungfish indicates that a Leu-enkephalin-coding gene, distinct from proenkephalin, must be expressed in lungfish. Potential candidates may include a prodynorphin- or other opioid-like gene. Furthermore, the absence of a Leu-enkephalin sequence in lungfish and amphibian proenkephalin would suggest that the mutations that yielded this opioid sequence in tetrapod proenkephalin occurred at some point in the radiation of the amniote vertebrates.     

Maturational changes in expression of enkephalin peptides in adrenal and extra-adrenal tissue of fetal and adult rabbits

Met-enkephalin immunoreactivity (MET-ENKi), total enkephalin immunoreactivity (TOTAL MET-ENKi) and catecholamines were measured in adrenal and extra-adrenal tissue of fetal, newborn and adult rabbits. Met-enkephalin peptides were detected in adrenal and extra-adrenal tissue by 29 days of gestation. There were progressive increases in TOTAL MET-ENKi in both the adrenal and extra-adrenal tissue during development. In 29-day-old fetuses, MET-ENKi represented 43 and 50% of the peptide content in adrenal and extra-adrenal tissues respectively. By 3 days after birth, MET-ENKi represented only 15 and 7% of the peptide content in the same tissues. In the adult adrenals, 10% of enkephalin peptides were found as MET-ENKi. There were progressive increases in adrenal and extra-adrenal catecholamine content in the fetal and newborn rabbits throughout development. The changes in the ratio of MET-ENKi to TOTAL MET-ENKi peptides suggest differences in posttranslational processing of proenkephalin peptide during maturation. We speculate that enkephalin peptides derived from proenkephalin A are important during fetal and early newborn life and that extra-adrenal tissue may be an important source of these peptides during development.     

Primate model of Parkinson''s disease: alterations in multiple opioid systems in the basal ganglia

A motor disorder similar to idiopathic Parkinson's Disease develops in rhesus monkeys after several daily repeated doses of N-methyl-4-phenyl, 1,2,3,6-tetrahydropyridine (MPTP). The concentrations of peptides derived from proenkephalin A, proenkephalin B, substance P and somatostatin were measured by specific radioimmunoassays in the basal ganglia of MPTP-treated monkeys. In MPTP-treated monkeys, dynorphin B concentration was reduced in the caudate. In the putamen, the concentrations of peptides derived from both proenkephalin A and proenkephalin B were decreased. In the globus pallidus, the concentrations of all opioid peptides tend to be increased, reaching significance only for alpha-neo-endorphin. In the substantia nigra, only Met-enkephalin concentration was reduced, while other peptides derived from either proenkephalin A or proenkephalin B were not changed. Substance P and somatostatin were not changed in any brain area examined. Some of the symptoms associated with Parkinson's Disease may be related to altered activity of endogenous opiates in basal ganglia.     

Ontogenesis of proenkephalin products in rat striatum and the inhibitory effects of low-level lead exposure

Certain developmental abnormalities have been associated with environmental exposure to lead and our previous studies have indicated that the endogenous opioid system is disrupted by this metal. In connection with this we report the ontogeny of proenkephalin products in the rat striatum determined by combined HPLC and bioassay and the effects of low-level lead exposure on this ontogeny. The development of Met-enkephalin levels was dissimilar from that of the other proenkephalin products, Met-enkephalyl-Arg6-Phe7, Met-enkephalyl-Arg6-Gly7-Leu8 and Leu-enkephalin. The ratios of Met-enkephalin containing peptides to Leu-enkephalin was less than the 6:1 ratio predicted from the proenkephalin structure. Lead (administered in the maternal drinking water, from conception to weaning at 100, 300 and 1000 ppm) caused a dose-related depression of the levels of proenkephalin products in rat striatum at 10, 21 and 30 days after birth. The most pronounced effects were observed at 10 days and the most persistent effects were seen with Met-enkephalin. Peak blood lead levels were below 45 micrograms/100 ml in the 100 and 300 ppm lead-dosed groups and in all lead-dosed groups at 10 days after birth. It is suggested that lead may have inhibitory effects on proenkephalin-processing enzymes.     

Differential response to a stress stimulus of proenkephalin peptide content in immune cells of naive and chronically stressed rats

Proenkephalin peptides produced by endocrine and nervous tissues are involved in stress-induced immunosuppression. However, the role of peptides produced by immune cells remains unknown. The present study examines the effect of acute and chronic foot-shock stress on proenkephalin peptide content in bone marrow (BMMC), thymus (TMC), and spleen (SMC) rat mononuclear cells. Proenkephalin was not processed to met-enkephalin in BMMC, while in TMC and SMC met-enkephalin represented 10% and 26% of total met-enkephalin-containing peptides, respectively. Naive rats receiving a stress stimulus showed a significant decrease of proenkephalin derived peptides in BMMC, TMC and SMC. However, in chronically stressed rats that already showed basal low peptide levels, a new stress stimulus produced a differential response in each immune tissue. That is, in BMMC peptide levels reached control rats values; in TMC remained unmodified; and in SMC, although precursors content increased, met-enkephalin levels were even lower than those observed in acutely stressed rats. Free synenkephalin content paralleled met-enkephalin changes in SMC of acutely and chronically stressed rats. The in vitro release of met-enkephalin and free synenkephalin increased in SMC of stressed rats. Met-enkephalin produced in SMC and partially processed proenkephalin peptides detected in BMMC, were only found in macrophages. However, met-enkephalin only appeared in bone marrow macrophages after at least 4 h of cell culture. Altogether, these results suggest that a stress stimulus induced proenkephalin peptide release from immune tissue macrophages. The differential response observed in chronically stressed rats suggest an alternative activation of heterogeneous proenkephalin-storing macrophage subpopulations.     

Opioid peptide release in the rat hippocampus after kainic acid-induced status epilepticus

It has been suggested that kainic acid enhances opioid peptide release. However, no direct evidence exists to support this hypothesis. The main aim of the present study was to determine whether such release occurs in the hippocampus of the rat after status epilepticus induced by kainic acid. Microdialysis experiments revealed significant opioid peptide release in the hippocampus 90-150 min (100%) and 270-300 min (50%) after kainic acid-induced status epilepticus. The peptides released were identified by high-performance liquid chromatography linked to radioimmunoassay as Met-enkephalin, Leu-enkephalin, Dynorphin-A (1-6), and Dynorphin-A (1-8). Reduced extracellular opioid peptide immunoreactivity was detected 28 days after status epilepticus (38% compared with control situation). The present results indicate an important activation of opioid peptide systems by kainic acid-induced status epilepticus. In addition, the reduced hippocampal extracellular opioid peptide levels long-term after kainic acid administration could have important implications for the progressive nature of epileptogenesis.     

Synenkephalin processing in embryonic rat brain

Synenkephalin (proenkephalin 1–70) is produced and secreted as an intact molecule or as a part of precursors in the adult brain and adrenal medulla, respectively. However, it is cleaved to low molecular weight peptides in proliferating immune cells. Considering that the pre-proenkephalin gene is expressed in the embryonic rat brain during the cell proliferation stage, we studied the processing of synenkephalin in embryonic rat brains (E18) and compared it with the processing in adult rat brains. IR-synenkephalin was measured by RIA using a C-terminally directed antiserum. Adult rat brains contained higher concentrations of immunoreactive (IR)-synenkephalin (2,612 + 264) than embryonic rat brain (1,361 + 100) (results in fmol/mg proteins, n = 5). Gel filtration chromatography (Sephadex G-50) showed that in the extracts of adult rat brain, 50% of the IR-synenkephalin eluted in the position of the authentic peptide (8 kDa) and the rest of the immunoreactivity corresponded to partially processed peptides of 4.0 and 2.5 kDa. In embryonic rat brains synenkephalin was processed to intermediate peptides of 2.5, 1.7 and mainly to a low molecular weight peptide of 1.0 kDa. The concentration of this last peptide, which was further characterized by affinity column and HPLC, represented 45% of the total immunoreactivity. IR-met-enkephalin in embryonic rat brains (analyzed before and after enzymatic digestion with trypsin and carboxypeptidase B) corresponded principally to non-processed or partially processed products. However, these were cleaved to free met-enkephalin in adult rat brains. These results indicate that the non-opioid portion of proenkephalin (synenkephalin-derived peptides) rather than the opioid portion (met-enkephalin-containing peptides) is fully cleaved to the low molecular weight peptides in the early stages of embryonic brain development, suggesting an involvement of synenkephalin products in nerve cell proliferation.     

Co-localization of proenkephalin mRNA using cRNA probes and a cell-type-specific immunocytochemical marker for intact astrocytes in vitro.

To determine whether cultured astrocytes express opioid gene mRNA, a method was developed for co-localizing a cell-type specific immunocytochemical marker for astrocytes, glial fibrillary acidic protein (GFAP), and proenkephalin mRNA in situ hybridization signal using high affinity cRNA probes. GFAP immunoreactivity and proenkephalin mRNA hybridization reaction were examined in intact glial cell preparations from neonatal mice that were cultured for 4-6 days prior to fixation. The double labelling method described herein permits the unambiguous identification of mRNA expression in specific populations of intact cultured cells using cell type-specific markers.     

Colocalization of immunoreactive proenkephalin and prodynorphin products in medullary neurons of the rat

This study addressed the possible coexistence of products of the proenkephalin and prodynorphin opioid peptide precursors in single neurons of the central nervous system of the rat. Antisera directed against met-enkephalin-arg-gly-leu and against Dyn B were used in immunohistochemical preparations of sections through the rat medulla. Examination of serial three micron frozen sections stained alternately with the two different antisera revealed that the majority of labelled neurons stain with only one of the two antisera. In specific area, however, immunoreactive m-enk and Dyn B could be detected in the same neuron. This was particularly true of the caudal ventrolateral nucleus of the solitary tract, where the two peptides were colocalized in most neurons. Other areas where the two peptides coexist include the midline raphe and the nucleus reticularis paragigantocellularis. These data provide the first evidence for colocalization of different opioid peptide families in single CNS neurons.     

Brain dynorphin and enkephalin systems in Fischer and Lewis rats: effects of morphine tolerance and withdrawal

Lewis rats are more likely to self-administer various drugs of abuse than Fischer rats. Here these two strains of rats were compared with regard to basal brain opioid peptide levels and the response to chronic morphine treatment and to naloxone-precipitated withdrawal. Lewis rats had lower basal dynorphin peptides in the substantia nogra, striatum (not Leu-enkephalinArg⁶) and VTA (not dynorphin B) and the pituitary gland. Leu-enkephalinArg⁶ levels were also lower in these structures (with the exception of striatum which had higher levels) and in the nucleus accumbens. There were also strain differences in the response to chronic morphine treatment; in the nucleus accumbens, morphine treatment increased dynorphin A levels in Fischer rats only, in the ventral tegmental area effects were opposite with increased dynorphin levels in Fischer and decreased levels in Lewis rats, in the hippocampus dynorphin levels were markedly reduced in Lewis rats only. In Fischer rats, chronic morphine strongly affected peptide levels in the substantia nigra and striatum, whereas Lewis rats responded less in these areas. Leu-enkephalin, which derives from both prodynorphin and proenkephalin, and Met-enkephalin, which derives from proenkephalin, were effected by chronic morphine mainly in Fischer rats, increasing levels in most of the brain areas examined. The results in this study show (1) strain differences in basal levels of prodynorphin-derived opioid peptides, (2) the prodynorphin system to be differently influenced by morphine in Lewis rats than in Fischer rats and 3) the proenkephalin system to be influenced by chronic morphine in brain areas related to reward processes only in Fischer rats.     

Proenkephalin processing enzyme with specificity toward paired basic residues purified from bovine adrenal chromaffin granules

A novel protease exhibiting substrate specificity toward paired basic residues has been partially purified from the soluble fraction of bovine adrenal chromaffin granules by utilizing an affinity chromatography on STI-Sepharose. The enzyme, with optimal pH around 7.5-9.5, is classified into a serine-protease by its inhibitor spectrum. The enzyme specifically cleaved the Lys-Arg bonds of two synthetic peptides containing the subsequence of proenkephalin A, but endogenous opioid peptides containing a single basic residue in the molecule [Met)enk-Arg-Phe, (Met)enk-Arg-Gly-Leu) were not affected by the enzyme. The unique substrate specificity of the enzyme, which is well in accord with the processing pattern of proenkephalin A in adrenal medulla, indicates that the enzyme may be physiologically involved in proenkephalin processing.     

Nicotine-induced alterations in peripheral tissue concentrations of native and cryptic Met- and Leu-enkephalin

This study examined the peripheral tissue distribution of native and cryptic Met- and Leu-enkephalin, and regulation of tissue enkephalins by nicotine. Met- and Leu-enkephalin concentrations showed widespread variation in tissue concentration and degree of processing. HPLC characterization of homogenate of spleen revealed that both native and cryptic immunoreactive Met-enkephalin are comprised of two peaks, one representing authentic Met-enkephalin pentapeptide and the other its sulfoxide. Subacute repeated administration of nicotine 0.1 mg/kg ip, six times at 30 min intervals, increased native Met- and Leu-enkephalin in adrenal medulla without affecting cryptic Met- and Leu-enkephalin concentrations, consistent with increased processing of larger peptides to Met- and Leu-enkephalin. Subacute nicotine decreased splenic concentrations of native and cryptic Met-enkephalin and native Leu-enkephalin, consistent with increased release of Met- and Leu-enkephalin from spleen and decreased synthesis of proenkephalin A or inadequate processing of larger peptides to enkephalin pentapeptides in spleen to compensate for the increased release during this period. HPLC characterization revealed that nicotine-induced decrease in native Met-enkephalin in spleen resulted from reductions in both pentapeptide and its sulfoxide. Nicotine also increased native Met-enkephalin in jejunum, decreased cryptic Met-enkephalin in heart atrium, increased native Leu-enkephalin in anterior pituitary and decreased cryptic Leu-enkephalin in jejunum. Nicotine may produce some of its effects through alterations in release of enkephalins from peripheral tissues.     

GABAergic regulation of striatal opioid gene expression.

Peptides derived from prodynorphin and preproenkephalin are located in GABAergic striatal projection neurons. We have used nucleic acid hybridization techniques to investigate the role of GABA in the regulation of striatal opioid peptide gene expression. Rats were treated with the GABA-transaminase inhibitors aminooxy acetic acid, ethanolamine O-sulphate and gamma-vinyl-GABA for one week. The GABA levels in the striatum were significantly elevated after each treatment. The GABA-transaminase-inhibitors decreased the striatal levels of the opioid peptides met-enkephalin and dynorphin(1-8) and concomitantly decreased the concentrations of the mRNAs coding for proenkephalin and prodynorphin. These findings indicate that GABA exerts an inhibitory influence on prodynorphin and proenkephalin gene expression in the striatum. The mechanisms underlying these inhibitions are discussed.     

Modulation of superoxide anion release from human polymorphonuclear cells by Met- and Leu-enkephalin

The present work describes the ability of Met- and Leu-enkephalin to modulate the superoxide anion (O2-) release from unstimulated human polymorphonuclear cells (PMN) and from PMN stimulated with phorbol myristate acetate (PMA). The direction (stimulation or suppression) and the magnitude of change were dependent upon the baseline reactivity of the donor's PMN. Both opioid peptides stimulated O2- release by PMN from donors with low baseline reactivity in a concentration-dependent manner. PMNs collected from donors with medium baseline reactivity incubated with Leu-enkephalin regardless of concentration released less O2- than control, nontreated PMNs. Met-enkephalin stimulated O2- release but only at 2 X 10(-15) M concentration. Superoxide anion release from PMNs of individuals with high baseline reactivity was concentration dependent and suppressed by Met- and Leu-enkephalin. Leu-enkephalin induced baseline reactivity was dependent upon progressive increase in the magnitude of change on O2- release (i.e., the higher the baseline the higher the magnitude of change in O2- generation). Met-enkephalin data show this also, but to a lesser extent. In cells stimulated with PMA, Met-enkephalin caused additional O2- release, while Leu-enkephalin was ineffective in triggering already stimulated cells. The modulating effect of both opioid peptides on superoxide anion release by human PMN is a short phenomenon that lasts up to 10 min after the addition of the peptide.     

The cloned μ, δ and κ receptors and their endogenous ligands: Evidence for two opioid peptide recognition cores

The opioid peptides are derived from three prohormone precursors referred to as proopiomelanocortin (POMC), proenkephalin (ProEnk) and prodynorphin (ProDyn). Following specific cleavage, several biologically active peptides are generated that can bind the μ, δ and κ receptors. The present study examines the receptor binding affinities of the POMC, ProEnk and ProDyn peptides to the cloned μ, δ and κ receptors expressed transiently in transfected COS-1 cells. Consistent with previous findings using brain homogenates, competition studies demonstrate that no opioid peptide family can be exclusively associated with a specific opioid receptor type. Short ProEnk peptides, such as Leu- and Met-enkephalin are selective for δ, but C-terminally extended peptides such as Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe have a high affinity to μ, δ and κ. Similarly, Peptide E, the BAM peptides, and metorphamide have a high affinity for all three opioid receptors types. While dynorphin A peptides and - and β-neoendorphin have a preference for κ, they also bind the cloned δ and μ receptors. Our findings do not easily fit a simple ‘message-address’ model where the Try-Gly-Gly-Phe core is extended and this gradually alters selectivity. Rather, the pattern appears more discontinuous, and would fit better with the idea of two similar but distinct cores; a Tyr-Gly-Gly-Phe Met- or Leu core that is necessary and sufficient for μ and δ but not κ and a Tyr-Gly-Gly-Phe-Met or Leu core with an Arg-X extension that is equally necessary and sufficient for κ.