Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity in the human gut

The presence of the proenkephalin A-derived peptide Met5-enkephalin-Arg6-Gly7-Leu8 was demonstrated throughout the human gastrointestinal tract. Highest concentrations of Met5-enkephalin-Arg6-Gly7-Leu8, as assessed by radioimmunoassay, were measured in the separated muscularis externa, while lower levels were found in the submucosa and only small amounts in the mucosa. The results are consistent with a neuronal location of this peptide in the human gut. Over 65% of total immunoreactivity coeluted with the authentic peptide in both molecular exclusion chromatography and HPLC, while most of the remainder activity eluted earlier on gel filtration. The latter material probably represents N-terminally extended Met5-enkephalin-Arg6-Gly7-Leu8. Taken together with previous studies, our results appear to indicate that there are important species differences in post-translational processing of proenkephalin A in gut nerves.     

Pro-enkephalin peptides possessing Met-enkephalin-Arg6-Gly7-Leu8-immunoreactivity in rat CSF, striatum and adrenal gland

Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae and extracts prepared from rat adrenal gland and striatum were subjected to Sephadex G-50 chromatography and HPLC. Fractions were monitored using specific radioimmunoassays (RIA) for the pentapeptide methionine enkephalin (Met-Enk) and methionine enkephalin-Arg6-Gly7-Leu8 (Met-EnkRGL). In rat CSF, striatum and adrenal gland, three Met-EnkRGL-immunoreactive (IR) peaks of Mrs 8000, 5000 and 1000 daltons were detected. The same peaks were also found to possess Met-Enk-immunoreactivity after enzyme digestion of Sephadex G-50 fractions with trypsin and carboxypeptidase B (CPB), suggesting their derivation from proenkephalin. HPLC of the 8K and 5K peaks on a column of Ultrapore RPSC showed them to elute discretely with similar retention times, indicative of hydrophobic peptides of large molecular weight. Their similar hydrophobicities yet significant separation during gel filtration would suggest that the 8K and 5K peptides are structurally closely related yet different with respect to their molecular weights. HPLC of the small molecular weight material from rat striatum and adrenal gland revealed the presence of Met-EnkRGL and Met-EnkRGL sulphoxide in both tissues. In rat striatum Met-Enk and its sulphoxide were also detected. The oxidised pentapeptide was found to be present in rat CSF, together with two unidentified small molecular weight Met-Enk-IR peaks detected without prior enzyme digestion of fractions. The small molecular weight Met-EnkRGL-IR material in rat CSF was found to be comprised of two unknown peptides which were less hydrophobic than Met-EnkRGL and its sulphoxide derivative.     

Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers in the major salivary glands of the rat: evidence for both sympathetic and parasympathetic origin

Summary The localization of the proenkephalin A-derived octapeptide, Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), was studied in the major salivary glands of Sprague-Dawley and Wistar rats with the indirect immunofluorescence method. MEAGL-immunoreactive nerve fibers were found around the acini, along intra-and interlobular salivary ducts and in close contact with blood vessels. In the parotid and submandibular glands tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibers around the acini, in association with intra- and interlobular salivary ducts and around blood vessels, while in the sublingual gland TH-immunoreactive nerve fibers were only seen around blood vessels. Parasympathetic neurons in submandibular ganglia contained MEAGL immunoreactivity. Moderate TH immunoreactivity was seen in some neurons of the submandibular ganglia. A subpopulation of sympathetic principal neurons in the superior cervical ganglion were immunoreactive for both MEAGL and TH. In the trigeminal ganglion, no MEAGL-immunoreactive sensory neurons or nerve fibers were observed. Superior cervical ganglionectomies resulted in a complete disappearance of TH-immunoreactive nerve fibers, while MEAGL-immunoreative nerve fibers were still present in the glands. The presence of MEAGL immunoreactivity in neurons of both sympathetic superior cervical ganglia and parasympathetic submandibular ganglia and the results of superior cervical ganglionectomies suggest, that MEAGL-immunoreactive nerve fibers in the major salivary glands of the rat have both sympathetic and parasympathetic origin.     

Specific polyclonal antibodies to the carboxyl terminus of [Met]enkephalin-Arg6-Gly7-Leu8

The octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu was recently isolated from bovine adrenal chromaffin granules and serves as a marker for proenkephalin from which it is derived. Polyclonal antisera which are highly specific for the carboxyl terminus have been raised against the synthetic peptide. The only significant cross-reactivity was with the 18.2-k Da and 5.3-k Da enkephalin-containing peptides (EC peptides) which contain the octapeptide at their carboxyl termini and the [des-Tyr] and [des-Tyr-Gly] congeners of the octapeptide. Extracts of bovine adrenal medulla and rat spinal cord were shown to contain significant amounts of the octapeptide, the two larger EC peptides, and the two smaller congeners.     

Localization of Met-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the gastrointestinal tract of rat and pig

One of the opioid precursor molecules, pre-pro-enkephalin A, contains within it, in addition to Leu-enkephalin (Leu-Enk) and Met-enkephalin (Met-Enk), Met-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-8), which is specific to this precursor. This study deals with the localization of Met-Enk-8-like immunoreactivity in the gastrointestinal tract of rat and pig. Immunoreactivity was identified in intramural nerve elements of rat and pig, and in gut endocrine cells of pig. Immunoreactive (IR) nerve fibers were seen mainly in the myenteric plexus of rat and in both the myenteric and submucosal plexuses of pig. Some IR fibers were dispersed throughout the lamina propria mucosae of rat. Porcine IR endocrine cells were dispersed in the epithelium from the pyloric antrum to the ileum, existing concomitantly with enterochromaffin (EC) cells. Specificity tests revealed that immunoreactivity to Met-Enk-8 antiserum was not influenced by preincubation of the antiserum with Leu-Enk and Met-Enk. This suggests the possibility that pre-pro-enkephalin A is contained in the gastroenteric nerves of rat and pig and in a population of porcine EC cells.     

Distribution and functional significance of Met-enkephalin-Arg6-Phe7-and Met-enkephalin-Arg6-Gly7-Leu8-like peptides in the blowflyCalliphora vomitoria

Summary Neuronal pathways in the retrocerebral complex and thoracico-abdominal ganglionic mass of the blowflyCalliphora vomitoria have been identified immunocytochemically with antisera against the extended-enkephalins, Met-enkephalin-Arg⁶-Phe⁷ (Met-7) and Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8). Neurons of the hypocerebral ganglion, immunoreactive to Met-8, have axons in the crop duct nerve and terminals in muscles of the crop and its duct. Certain neurons of the hypocerebral ganglion are also immunoreactive to Met-7, and axons from these cells innervate the heart. Met-8 immunoreactive nerve terminals invest the cells of the corpus allatum. The source of this material is believed to ve a single pair of lateral neurosecretory cells in the brain. There is no Met-7 immunoreactive material in the corpus allatum. In the corpus cardiacum neither Met-7 nor Met-8 immunoreactivity is present in the cells. However, in the neuropil of the gland certain fibres, with their origins elsewhere, do contain Met-8 immunoreactivity. The most prominent neurons in the thoracic ganglion are the Met-7 immunoreactive ventral thoracic neurosecretory cells, axons from which project to neurohaemal areas in the dorsal neural sheath and also, via the ventral connective, to the brain. Co-localisation studies show that the perikarya of these cells are immunoreactive to antisera raised against several vertebrate-type peptides, such as Met-7, gastrin/cholecystokinin and pancreatic polypeptide. However, their axons and terminals show varying amounts of the peptides, suggesting differential transport and utilisation. Only a few cells in the thoracic ganglion are immunoreactive to Met-8 antisera. These lie close to the nerve bundles suppling the legs. In the abdominal ganglion, Met-8 immunoreactive neurons project to the muscles of the hindgut. This study suggests that the extended enkephalin-like peptides ofCalliphora may have a variety of different roles: as neurotransmitter or neuromodulator substances; in the direct innervation of effector organs; and as neurohormones.     

Coexistence of catecholamine and methionine enkephalin-Arg6-Gly7-Leu8 in neurons of the rat ventrolateral medulla oblongata

Summary An overlapping distribution of catecholamine-containing cells and proenkephaline—A derived peptide-containing neurons have been identified in the rat medulla oblongata. However, it is not evident whether the coexistence of these bioactive substances occurs in the same neurons or not. Therefore, we examined the coexistence of catecholamine and methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), a proenkephaline—A derived peptide, using a combination of histofluorescence and peroxidase-anti-peroxidase (PAP) immunohistochemical (modified formaldehyde-glutalaldehyde (Faglu)) methods on the same tissue sections. We found one third of A1/C1 catecholamine fluorescent cells show MEAGL-like immunoreactivity.     

Distribution and functional significance of Met-enkephalin-Arg6-Phe7- and Met-enkephalin-Arg6-Gly7-Leu8-like peptides in the blowfly Calliphora vomitoria

Summary Neuronal pathways immunoreactive to antisera against the extended-enkephalins, Met-enkephalin-Arg⁶-Phe⁷ (Met-7) and Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8), have been identified in the brain of the blowfly Calliphora vomitoria. Co-localisation with other enkephalins in certain neurons suggests that a precursor similar to preproenkephalin A exists in insects and that differential enzymatic processing occurs as in vertebrates. Co-localisations of the extended-enkephalin-like peptides with other vertebrate-type peptides, including cholecystokinin and pancreatic polypeptide, also occur. The enkephalinergic pathways are specific, comprising a few groups of highly characteristic neurons and areas of neuropil. Of special interest is the finding that parts of the antennal chemosensory and the optic lobe visual systems contain Met-8 immunoreactive neurons. Within the median neurosecretory cell groups, some of the giant neurons show immunoreactivity to Met-8 and others to both Met-8 and Met-7. Fibres from these cells project to the corpus cardiacum and also to the suboesophageal ganglion, where arborisations occur in the tritocerebral neuropil. Co-localisation studies of these cells have shown that at certain terminals, one particular type of peptide is the dominant neuroregulator, whilst at other terminals, within the same cell, a different co-synthesised peptide predominates. Several groups of lateral neurosecretory cells show clearly defined enkephalinergic pathways, most of which have connections with the central body. The complex patterns of immunoreactivity seen in terminals in the different parts of the central body, suggest an important role for the enkephalin-like peptides in the integration of multimodal sensory inputs. The physiological functions of the extended-enkephalin-like peptides in the brain of Calliphora is still unknown, but the anatomical evidence suggests they may have a role similar to that in mammals, where they are thought to control aspects of feeding behaviour.     

Cardiovascular activities of intravenous methionine-enkephalin-Arg6-Phe7 and methionine-enkephalin-Arg6-Gly7-Leu8 in the conscious dog

The preproenkephalin A molecule from the adrenal medulla contains the opioid peptides methionine-enkephalin (Met-ENK), leucine-enkephalin (Leu-ENK), methionine-enkephalin-Arg6-Phe7 (heptapeptide), and methionine-enkephalin-Arg6-Gly7-Leu8 (octapeptide). In the conscious, chronically instrumented dog, Met-ENK and Leu-ENK simultaneously increase heart rate and systemic arterial pressure following intravenous administration. In 19 of 23 dogs, heptapeptide produced a response identical to Met-ENK and Leu-ENK, which was inhibited by naloxone but unaffected by the dipeptidyl carboxypeptidase inhibitor SQ20881. However, in four dogs, heptapeptide produced only a fall in systemic pressure associated with an increase in heart rate despite characteristic Met-ENK responses in the same dogs; naloxone did not appear to alter this hypotensive response. Octapeptide produced slight increases in systemic pressure and heart rate. These data suggest that heptapeptide may possess intrinsic cardiovascular activity at opiate receptors; however, in certain dogs, non-opiate mechanisms, perhaps histamine release, may predominate.     

Proteolytic conversion of [Met]enkephalin-Arg6-Gly7-Leu8 by brain synaptic membranes. Characterization of formed peptides and mechanism of proteolysis

The degradation of the enkephalin-containing octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu (YGGFMRGL) was systematically investigated by incubating the peptide with synaptic membranes from rat striatum or with purified peptidases. The degradation products were derivatized with 4-dimethylamino-azobenzene-4'-isothiocyanate and then analyzed by high pressure liquid chromatography and by amino-terminal analysis. The incubation of YGGFMRGL with synaptic membranes yielded YGG, YGGF, YGGFM, and MR in a manner that was linear with respect to time. The corresponding carboxyl-terminal fragments FMRGL, MRGL, and RGL could not be detected, which suggests that the degradation of YGGFMRGL by synaptic membranes occurs by carboxypeptidase activity. The incubation of YGGFMRGL with different purified peptidases produced cleavage patterns unique from that seen with synaptic membranes. Enkephalinase recognized only the Gly-Phe bond to produce YGG and FMRGL. Thermolysin recognized the Gly-Phe bond and the Phe-Met bond to yield YGG, YGGF, FMRGL, and MRGL. Angiotensin-converting enzyme (ACE) produced primarily YGGF, MR, and lesser amounts of YGGFMR and YG. The formation of YGG, YGGF, and YGGFM by synaptic membranes could be stimulated 3-fold by the addition of 30 mM NaCl and inhibited by MK-422, an ACE inhibitor, with an IC50 of 3 nM. These data suggest that ACE, a dipeptidyl carboxypeptidase, is the primary enzyme involved in the degradation of YGGFMRGL in brain. ACE apparently works in concert with another carboxypeptidase in brain to yield YGGFM and YGG since the carboxyl-terminal peptides RGL and FMRGL could not be detected.     

Occurrence of met-enkephalin,met-enkephalin-Arg6-Phe7 and met-enkephalin-Arg6-Gly7-Leu8 in gastrin cells of hog antral mucosa

Summary Region-specific antisera to three enkephalins: met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸, together with four region specific antisera to progastrin: C-terminal G17 specific, N-terminal G34 specific, cryptic peptides A- and B-specific, were used in immunohistochemical studies of hog antral mucosa. A sub-population (6–10%) of the gastrin-containing endocrine cells (G-cells) was found to react with antisera to met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸. About 30% of all the enkephalin-containing cells were identified as G-cells. The results indicate that a fraction of G-cells produces both enkephalin-like peptides and gastrin.     

Receptor binding and G-protein activation by new Met5-enkephalin-Arg6-Phe7 derived peptides

Met5-enkephalin-Arg6-Phe7 (Tyr-Gly-Gly-Phe-Met-Arg-Phe, MERF) is a naturally occurring heptapeptide that binds to opioid and non-opioid recognition sites in the central nervous system. Four synthetic analogs with single or double amino acid substitutions were prepared by solid phase peptide synthesis to achieve proteolytically more stable structures: Tyr-D-Ala-Gly-Phe-Met-Arg-Phe (I), Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (II), Tyr-D-Ala-Gly-Phe-L-Nle-Arg-Phe (III) and Tyr-Gly-Gly-Phe-L-Nle-Arg-Phe (IV). In this study receptor binding characteristics and G-protein activation of MERF and its derivatives were compared in crude membrane fractions of frog and rat brain. Synthetic MERF-derived peptides were potent competitors for [3H]MERF and [3H]naloxone binding sites with the exception of analog (II) which turned to be substantially less active. The presence of 100 mM NaCl or 100 microM 5'-guanylylimidodiphosphate, Gpp(NH)p, decreased the affinity of the peptides in [3H]naloxone binding assays, suggesting that these ligands might act as agonists at the opioid receptors. Some of the compounds were also used to stimulate guanosine-5'-O-(3-[gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding in rat and frog brain membranes at concentrations of 10(-9)-10(-5) M. The EC50 values of analog (II) were the highest in both tissues. Analog (I) was as effective as MERF in rat brain membranes, but showed lower maximal stimulation in frog brain preparation. Again, analog (II) seemed to be the least efficacious peptide that stimulated [35S]GTPgammaS binding only by 59%. Specificity of the peptides was further investigated by the inhibition of agonist-stimulated [35S]GTPgammaS binding in the presence of selective antagonists for the opioid receptor types. The mu-selective antagonist cyprodime displayed the lowest potency in inhibiting the effects of the peptides, whereas norbinaltorphimine (kappa-selective antagonist) and naltrindole (delta-selective antagonist) were quite potent in both tissues. We concluded that MERF and its derivatives are able to activate G-proteins mainly via kappa- and delta-opioid receptors.     

Immunohistochemical localization of Met-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8, Met-enkephalin-Arg6-Phe7 and Leu-enkephalin in human adrenal medulla and pheochromocytomas

Immunohistochemical localization of Met-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8, Met-enkephalin-Arg6-Phe7 and Leu-enkephalin was studied in human adrenal medulla and pheochromocytomas at the light and electron microscopic levels. Both adrenal medulla and pheochromocytomas (4 adrenal, 1 extra-adrenal) showed scattered or clustered cells which contained all of the above peptides and suggested the production of proenkephalin A. The presence of these peptides predominantly in the secretory granules suggested that proenkephalin A is processed to final products mainly in the secretory granules. The localization of Met-enkephalin-Arg6-Gly7-Leu8 and Met-enkephalin-Arg6-Phe7 in cisternae of rough endoplasmic reticula indicated their actual production in pheochromocytomas.     

Age-related and diurnal changes in Met5-Enk-Arg6-Phe7 and Met5-enkephalin contents of pituitary and rat brain structures

The beta-endorphin, met5-enkephalin-arg6-phe7 (MEAP) and met5-enkephalin (ME) changes related to age and diurnal rhythms were studied in various regions of rat brain and in the pituitary by specific radioimmunoassays. The contents of MEAP, met5-enkephalin and beta-endorphin were higher in the pituitary of old rats (18 months old) than that of young rats (23 days old) while the content of these opioid peptides was higher in the hypothalamus of young rats than in that of old rats. Beta-endorphin was also higher in the striatum of 23 days old rats, but no age-associated changes were observed in the hippocampus, brain stem or cortex. In the diurnal rhythm study, it was found that in the hypothalamus and striatum of the adult rat (2-3 months old), both MEAP and ME contents were higher at mid-dark than at mid-light and that in the intermediate posterior lobe of the pituitary, the ME content was also higher at mid-dark.     

The action of a synthetic derivative of Met5-enkephalin-Arg6-Phe7 on behavioral and endocrine responses

The neuroendocrine and behavioral effects of Tyr-d-Ala-Gly-Phe-d-Nle-Arg-Phe (DADN), a more stable derivative of the endogenous opiate Met-enkephalin related peptide Met⁵-enkephalin-Arg⁶-Phe⁷ were investigated in mice. The behavioral experiments consisted of monitoring the horizontal (square crossing) and vertical (rearing) locomotion in the open field system. To evaluate the effect of the heptapeptide on the hypothalamo-pituitary-adrenal (HPA) axis, the plasma corticosterone level was measured. DADN induced dose-dependent increases in locomotion and rearing 30 min after intracerebroventricular injection and also elicited marked activation of the hormonal stress response. To elucidate the receptors involved in the mediation of these actions, animals were pretreated with the nonselective opioid antagonist naloxone, the selective κ-receptor antagonist nor-binaltorphimine or the μ₁-receptor blocker naloxonazine. Both the HPA activation and the behavioral responses were diminished by the preadministration of naloxone. Nor-binaltorphimine did not display a significant effect, while naloxonazine completely abolished the hyperactivity and the corticosterone elevation elicited by the analog. These findings suggest that μ-receptors predominate in the mediation of the neuroendocrine actions of DADN, while κ-receptors do not play a significant role.     

Coexistence of catecholamine and methionine enkephalin-Arg6-Gly7-Leu8 in neurons of the rat ventrolateral medulla oblongata. Application of combined peptide immunocytochemistry and histofluorescence method in the same vibratome section

An overlapping distribution of catecholamine-containing cells and proenkephaline-A derived peptide-containing neurons have been identified in the rat medulla oblongata. However, it is not evident whether the coexistence of these bioactive substances occurs in the same neurons or not. Therefore, we examined the coexistence of catecholamine and methionine-enkephalin-Arg6-Gly7-Leu8 (MEAGL), a proenkephaline-A derived peptide, using a combination of histofluorescence and peroxidase-anti-peroxidase (PAP) immunohistochemical (modified formaldehyde-glutalaldehyde (Faglu)) methods on the same tissue sections. We found one third of A1/C1 catecholamine fluorescent cells show MEAGL-like immunoreactivity.     

N-linked glycosylation of a proenkephalin A-derived peptide. Evidence for the glycosylation of an NH2-terminally extended Met-enkephalin Arg6-Gly7-Leu8 variant

To investigate the possibility that the opioid peptide precursor proenkephalin A was glycosylated, we utilized an antiserum raised against the COOH terminus of Met-enkephalin Arg6-Gly7-Leu8 (MERGL) to identify and characterize enkephalin-containing peptides from extracts of bovine adrenal medulla. Sephadex G-50 gel filtration separated two immunoreactive peaks which had apparent masses of 9 and 6 kDa. Anion-exchange chromatography and reverse-phase high pressure liquid chromatography (HPLC) revealed that the 9-kDa material was a heterogenous mixture of immunoreactive peptides, of which one (9K-MERGL Ia) was purified to homogeneity. The 6-kDa material separated into two major immunoreactive peaks (6K-MERGL I and 6K-MERGL II) on anion-exchange chromatography, and these were obtained in an homogenous form after reverse-phase HPLC. Amino acid sequencing, together with immunological characterization, indicated that the three peptides were identical in chain length, and corresponded to proenkephalin A 116-165. They contained the sequence Asn-Ser-Ser which is a potential N-glycosylation site. In 9K-MERGL Ia, but not the others, automated Edman amino acid sequencing was unable to detect the relevant asparagine residue, suggesting that this residue has been chemically modified. Further investigation of the 9K-MERGL material using lectin affinity chromatography provided direct evidence of glycosylation. Verification of this result was obtained using the specific enzyme glycopeptidase F (glycopeptide-N-glycosidase) which demonstrated that 9K-MERGL contained, in part, N-linked oligosaccharide chains. These results show that an NH2 terminally extended Met-enkephalin Arg6-Gly7-Leu8 variant was N-glycosylated, and hence indicate that the precursor polypeptide proenkephalin A can be glycosylated during translation in the rough endoplasmic reticulum.     

Immunohistochemical and biochemical evidence for the presence of the pentapeptide met-enkephalin and the heptapeptide met-enkephalin-Arg6-Phe7 but not the octapeptide met-enkephalin-Arg6-Gly7-Leu8 in amphibian chromaffin cells

Using specific antibodies to met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸, we have studied the distribution of these opioid peptides in the frog adrenal gland by means of the indirect immunofluorescence technique. Bright staining of all chromaffin cells was observed by application of met-enkephalin and met-enkephalin-Arg⁶-Phe⁷ antisera. No nerve endings could be detected. A few chromaffin cells were weakly stained by met-enkephalin-Arg⁶-Gly⁷-Leu⁸ antiserum. Using a specific radioimmunoassay for met-enkephalin, the dilution curve of frog adrenal extracts was parallel to that of synthetic met-enkephalin. The concentration of met-enkephalin-like material measured in crude acetic extracts of frog adrenals (2.31 ± 0.16 pmol/mg w. wt) was high when compared to those reported for most mammalian species. No leu-enkephalin and virtually no met-enkephalin-Arg⁶-Gly⁷-Leu⁸ were detected by the corresponding radioimmunoassays. Reverse phase HPLC analysis revealed that oxidized met-enkephalin was the main form of met-enkephalin detected in acetic extracts of frog adrenals. HPLC separation showed the presence of a peptide co-eluting with synthetic met-enkephalin-Arg⁶-Phe⁷. Higher molecular weight forms were also separated by HPLC. These results show the presence of both met-enkephalin and the heptapeptide met-enkephalin-Arg⁶-Phe⁷ and the lack of met-enkephalin-Arg⁶-Gly⁷-Leu⁸ in frog chromaffin cells.     

Immunohistochemical studies for multiple peptide-immunoreactivities and co-localization of Met-enkephalin-Arg6-Gly7-Leu8, neuropeptide Y and somatostatin in human adrenal medulla and pheochromocytomas

Three normal human adult adrenal medullas and 12 cases of pheochromocytomas were studied for immunohistochemical localization of various peptides. Met-enkephalin-Arg6-Gly7-Leu8 (MEAGL) was present in all cases of pheochromocytomas. The normal adrenal medulla showed cells immunoreactive for MEAGL, neuropeptide tyrosine (NPY) and proopiomelanocortin derived N-terminal fragment (NTF). MEAGL and NPY were co-localized in some adrenal medullary cells. Pheochromocytomas showed striking multiple immunoreactivities regardless of histologic types, pleomorphic or organoid. Ten cases showed immunoreactivities for more than two peptides. All cases showed immunoreactivity for MEAGL and 9 cases showed NPY positive cells. Some tumor cells contain both MEAGL and NPY in the cytoplasm. Six cases were positive for somatostatin. Some tumor cells were shown to contain both MEAGL and SS. The appearance of SS and other peptides was considered to be related to the neoplastic transformation of the adrenal medulla.