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.     

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.     

Distribution of Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactivity in the rat and mouse pituitary gland.

The distribution of the octapeptide Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL), a proenkephalin A-derived opioid peptide, in the rat and mouse pituitary gland was studied using the indirect immunofluorescence technique and immunoelectron microscopy. The anterior lobe contained a few MEAGL-immunoreactive cells but no nerve fibers. A previously unknown enkephalin-immunoreactive nerve fiber system was revealed in the intermediate lobe. These fibers originated in a dense MEAGL-immunoreactive plexus located along the border between the intermediate and posterior lobes and were distributed throughout the lobe. In the posterior lobe, MEAGL immunoreactivity was found in a very dense network of varicose fibers that was evenly distributed over the entire lobe. These results provide a morphological correlate for previous chemical studies and together with them suggest that MEAGL-immunoreactive innervation regulates endocrine functions of the intermediate and posterior lobes directly at the pituitary level.     

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.     

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.     

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.     

Maturation of an NH2-terminally extended thermostable alpha-amylase in Bacillus subtilis: a possible mechanism examined by in vitro experiments.

An artificially inserted extra peptide (21 amino acid peptide) between the B. subtilis alpha-amylase signal peptide and the mature thermostable alpha-amylase was completely cleaved by B. subtilis alkaline protease in vitro. The cleavage to form a mature enzyme was observed between pH 7.5 and 10, but not between pH 6.0 and 6.5, although a similar protease activity toward Azocall was observed between pH 6.0 and 7.5. To analyze the effects of pH on the cleavage, CD spectra at pH 6, 8, and 11 of the NH2-terminally extended thermostable alpha-amylase were analyzed and the results were compared with those of the mature form of the alpha-amylase. It is suggested that the cleavage of the NH2-terminally extended peptide is controlled by the secondary and tertiary structure of the precursor enzyme. Similar cleavage of different NH2-terminally extended peptides by the alkaline protease was also found in other hybrid thermostable alpha-amylases obtained.     

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.     

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.     

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.     

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.     

Characterisation of N-terminally extended met-enkephalin Arg6Gly7Leu8 variants in the porcine upper digestive tract

Proenkephalin A-derived peptides are known to occur in the gut, but their precise identity is uncertain. We report here the isolation of N-terminally extended forms of Met-enkephalin Arg6Gly7Leu8 from porcine upper digestive tract monitored by radioimmunoassay. A single major form was identified in pyloric antral muscle and mucosa, but in the duodenum two major forms were detected. Microsequence analysis together with immunological data revealed that the antral mucosal peptide and the most acidic duodenal peptide had identical amino-acid sequences, corresponding to a 5.3 kDa peptide terminating in Met-enkephalin Arg6Gly7Leu8. The data indicate that high-molecular-weight peptides may constitute a major proportion of gut opioid peptide immunoactivity.     

The ADP/ATP carrier is the 32-kilodalton receptor for an NH2-terminally myristylated src peptide but not for pp60src polypeptide.

Membrane binding of pp60src is initiated via its myristylated NH2 terminus. To identify a candidate pp60src docking protein or receptor in the membrane, a radiolabelled peptide corresponding to the pp60src NH2-terminal membrane binding domain was cross-linked to fibroblast membranes and found to specifically label a 32-kDa protein. This protein was purified by appending an affinity tag to the peptide probe so that the cross-linked complex could be isolated via affinity chromatography. Microsequencing indicated that the 32-kDa protein was the mitochondrial ADP/ATP carrier (AAC). This result was further confirmed by the ability of an antibody to the AAC to immunoprecipitate the cross-linked complex, by the ability of certain inhibitors of the AAC to block cross-linking, and by membrane fractionation to show that complex formation occurred essentially exclusively in the mitochondrial fraction. While the AAC bound the myristyl-src peptide in a specific manner both in vitro and in vivo, its localization to the inner membrane of the mitochondrion precludes its being a pp60src binding protein. An analysis of pp60v-src binding in vitro was consistent with this expectation. Thus, use of a myristyl-src peptide revealed an unexpected and previously unidentified binding capacity of the AAC, most likely related to the ability of long-chain fatty acyl coenzyme As to serve as AAC inhibitors. The amphipathic nature of the pp60src NH2 terminus suggests alternative strategies for uncovering pp60src membrane binding species.     

Immunocytochemical mapping of neuronal pathways from brain to corpora cardiaca/corpora allata in the cockroach Diploptera punctata with antisera against Met-enkephalin-Arg6-Gly7-Leu8

Summary Neuronal circuits in the brain and retrocerebral complex of the cockroach Diploptera punctata have been mapped immunocytochemically with antisera directed against the extended enkephalin, Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8). The pathways link median and lateral neurosecretory cells with the corpus cardiacum/corpus allatum complex. In females, nerve fibres penetrate the corpora allata and varicosities or terminals, immunoreactive to Met-8, surround the glandular cells. Males differ in having almost no Met-8 immunoreactivity in the corpora allata. The corpora cardiaca of both males and females are richly supplied with Met-8 immunoreactive material, in particular in the cap regions immediately adjacent to the corpora allata. A similarity in the amino-acid sequences of Met-8 and the C-terminus of the recently characterised allatostatins of D. punctata suggests that the pathways identified with the Met-8 antisera may be the same as those by which the allatostatins are transported from the brain to the corpus allatum. In comparative studies on the blowfly Calliphora vomitoria, similar neuronal pathways have been identified except that no sexual dimophism with respect to amounts of immunoreactive material within the corpus allatum has been observed. These results suggest a possible homology in the neuropeptide regulation of the gland.     

Heterodetic bicyclic decapeptide cyclo (Glu1-Leu2-Pro3-Gly4-Ser5-Ile6-Pro7- Ala8) cyclo-(1 gamma-5 beta) Phe9-Gly10. Synthesis and 2-D NMR conformational analysis

Bicyclic peptides are useful model molecules that can mimic the constrained local folding of a great number of natural peptides and proteins, such as ionophoric peptides, enzyme active site, and ligand-receptor active site. The synthesis of the bicyclic title compound with the liquid phase method is described with experimental details. Of particular interest is the heterodetic closure of the second ring. The peptide showed a complexing activity with metal cations like Ba2+, Ca2+, and Mg2+. The free bicyclic peptide conformation in solution has been studied by means of NMR spectroscopy and a plausible structure model worked out with model building on NMR constraints is proposed.     

Evidence for an extended 7SL RNA structure in the signal recognition particle.

The signal recognition particle (SRP) functions in conjunction with the SRP receptor to target nascent ectoplasmic proteins to the protein translocation machinery of the endoplasmic reticulum membrane. SRP is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of 7SL RNA 300 nucleotides long. SRP has previously been visualized by a variety of electron microscopic techniques as a rod-shaped particle 24 nm long and 6 nm wide. We report here microanalysis by electron spectroscopic imaging which localizes the RNA molecule in SRP to primarily the two ends of the particle. These results suggest that the single 7SL RNA molecule spans the length of the particle. Micrographs from a scanning transmission electron microscope permit visualization of unstained SRP with low electron exposure, as well as the direct measurement of the mol. wt of the particle. These micrographs confirm our earlier suggestion that SRP is divided into three structural domains and allow discrimination of the two ends of the structure. The results of both techniques have been combined in a model for the structure of SRP in which we propose the basic orientation of the 7SL RNA. The structure proposed is consistent with the secondary structure predicted for the RNA and with biochemical data.     

Bicyclic peptides: solution conformation and Ca2+ binding of the heterodetic bicyclic decapeptide cyclo(Glu1-Leu2-Pro3-Gly4-Ser5-Ile6-Pro7-Ala8)- cyclo(1 gamma----5 beta)Phe9-Gly10

The conformational behavior of a heterodetic bicyclic decapeptide (BCPLT) in the absence and in the presence of calcium ions has been studied by means of mono and two-dimensional nmr techniques. Free BCPLT possesses a quite compact structure stabilized by intramolecular bonds and turns. In the structure a cluster of carbonyls is located in a cavity that is supposed to be the cation binding site.