Existence of Met-enkephalin-Arg6-Gly7-Leu8 with Met-enkephalin,Leu-enkephalin and Met-enkephalin-Arg6-Phe7 in the brain of guinea pig,rat and golden hamster

High performance liquid chromatography (HPLC) coupled with specific radioimmunoassays for methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-E-Arg⁶-Gly⁷-Leu⁸), methionine-enkephalin (Met-E), leucine-enkephalin (Leu-E) and methionine-enkephalin-Arg⁶-Phe⁷ (Met-E-Arg⁶-Phe⁷) has demonstrated that Met-E-Arg⁶-Gly⁷-Leu⁸ exists together with Met-E, Leu-E and Met-E-Arg⁶-Phe⁷ in the brain of guinea pig, rat and golden hamster. The content of Met-E-Arg⁶-Gly⁷-Leu⁸ was comparable to those of Leu-E and Met-E-Arg⁶-Phe⁷, whereas that of Met-E was the highest among the four opioid peptides. These results are compatible with the recent studies on the nucleotide sequence of cloned cDNA for preproenkephalin from bovine adrenal medulla, which reveal that this precursor molecule contains four copies of Met-E and one copy each of Leu-E, Met-E-Arg⁶-Phe⁷ and Met-E-Arg⁶-Gly⁷-Leu⁸. The co-existence of Met-E-Arg⁶-Gly⁷-Leu⁸ with Met-E, Leu-E and Met-E-Arg⁶-Phe⁷ suggests that their biosynthetic pathway in the brain is similar to that in the adrenal medulla.     

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.     

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.     

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.     

Substance P degrading systems of rat parotid and hypothalamus

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu⁶]substance P6–11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K⁺ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu⁶]substance P6–11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe⁸-Gly⁹ with minor cleavage sites between Phe⁷-Phe⁸ and Gly⁹-Leu¹⁰. In the rat parotid slice system the major cleavage occurs between Gly⁹-Leu¹⁰ with a minor cleavage site between Phe⁷-Phe⁸. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH₂, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.     

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.     

Nmr and computer-aided studies of the three interchanging stereoisomers of the cyclic hexapeptide cyclo[-Pro1-Gly2-Glu3(OBzl)-Pro4-Phe5-Leu6-]: Unexpected observation of a cis isomer of a secondary amide peptide bond in the presence of two trans proline peptide bonds

The cyclic hexapeptide cyclo[-Pro¹-Gly²-Glu³(OBzl)-Pro⁴-Phe⁵-Leu⁶-] ( 1 ; OBzl: benzyl ester) was modeled and synthesized to be used as a chiral site for the separation of enantiomers. Total correlation spectroscopy and nuclear Ovehauser effect spectroscopy spectra of the peptide in CDCl₃ showed the presence of three stereoisomers. The two dominant stereoisomers 1a and 1b exchanged chemically with each other, while the minor stereoisomer 1c exchanged exclusively with the stereoisomer 1b . Stereoisomer 1a had two cis proline peptide bonds while stereoisomer 1b had all-trans peptide bonds. The stereoisomer 1c had, for nonstrained peptides, an unusual cis phenylalanine peptide bond while both proline peptide bonds were trans. © 1993 John Wiley & Sons, Inc.     

Conformation of CD4-derived cyclic hexapeptides by nmr and molecular dynamics

Two cyclic hexapeptides, cyclo[Ala¹-D -Ala²-Ser³-Phe⁴-Gly⁵-Ser⁶] and cyclo[Ala¹-Gly²-Ser³-Phe⁴-Gly⁵-Ser⁶], derived from the loop portion of the C′C″ ridge of CD4, were characterized by high-resolution nmr spectroscopy and simulated annealing studies. In DMSO-d₆ both of these peptides display a single conformer on the nmr time scale with two intramolecular H-bond (1 ← 4) stabilized β-turns at positions 2–3 and 5–6. The nmr derived distance constraints were used in simulated annealing calculations to generate the solution structures. These structures adopt energetically comparable conformational substates that are not resolvable on the nmr time scale. In aqueous solution, the H-bond stabilized β-turn conformation for cyclo [Ala-D -Ala-Ser-Phe-Gly-Ser] is no longer the predominant structural form. Structures generated using molecular dynamics simulations with no experimental constraints were compared with those from nmr analysis. The correlation between these two sets of structures allows the use of molecular simulations as a predictive tool for the conformational analysis of small peptides. © 1994 John Wiley & Sons, Inc.     

The preference of putative pro-enkephalins for different types of opiate receptors

Certain endogenous opioid peptides, which structurally resemble potential enkephalin precursors (pro-enkephalins), were tested for their interaction with μ-, δ- and κ-type of opiate receptors. These studies employed the technique of the development of tolerance towards selective opioid agonists on the isolated guinea-pig ileum and mouse vas deferens preparations. Additionally, binding studies in rat brain homogenate were performed to determine the affinity of these compounds towards different receptor sites in the CNS. These investigations revealed that dynorphin_1–13 and α-neo-endorphin_1–8 may preferentially interact with the κ-type of opiate receptors, dynorphin_1–9 exhibits both δ- and κ-agonistic activity and met-enkephalin-Arg⁶-Phe⁷ is a ligand with selectivity for δ-receptors.     

Distribution of neuropeptides in the porcine stellate ganglion

The localization and distribution of neuropeptides including neuropeptide Y (NPY), [Met⁵]enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P and somatostatin (SOM) were analyzed in the stellate ganglion of the pig by use of the indirect immunofluorescence technique. NPY, MEAGL, SOM, VIP and CGRP immunoreactivities were found to exist in subpopulations of neuronal cell bodies of the stellate ganglion. A population of the small intensely fluorescent (SIF) cells showed MEAGL immunoreactivity. In addition, the presence of NPY-, MEAGL-, CGRP-, SP-, SOM- and VIP-immunoreactive nerve fibers and axonal varicosities were observed in the stellate ganglion. The localization and pattern of distribution of these peptides in the porcine stellate ganglion were compared with studies carried out on stellate ganglia of other mammalian species.