Immunocytochemical analysis of proenkephalin-derived peptides in the amphibian hypothalamus and optic tectum

[Met5]-Enkephalin-, [Met5]-enkephalin-Arg6-, [Met5]-enkephalin-Arg6-Phe7-, metorphamide- and BAM 22P-like peptides could be localized in the amphibian brain by immunocytochemistry. However, a [Met5]-enkephalin-Arg6-Gly7-Leu8-like peptide could not be detected in the brain of any anuran species with an antiserum that was capable of detecting this octapeptide in mammalian brain. A synenkephalin-like peptide also could not be detected in the anuran brain with an antiserum that was capable of detecting the antigen in bovine and porcine brain. Although the intensity of proenkephalin-like immunoreactivity depended on the antiserum used, its distribution appeared to be identical with all of the effective antisera. Antisera directed against somatostatin and corticotropin-releasing factor stained perikarya, nerve fibers and terminals in the anuran brain with a distribution that was different from antisera directed against proenkephalin-derived peptides. The distribution of proenkephalin-containing perikarya and nerve fibers in the regions of the anuran brain selected for study showed many similarities to the distribution of proenkephalin-containing perikarya and nerve fibers in the same regions of the amniote brain.     

The opioid octapeptide Met5-enkephalin-Arg6-Gly7-Leu8: characterization and distribution in rat spinal cord

The regional quantitation, immunohistochemical localization and molecular heterogeneity of Met5-enkephalin-Arg6-Gly7-Leu8 were examined in rat spinal cord with a specific radioimmunoassay. A rostrocaudal gradient in Met5-enkephalin-Arg6-Gly7-Leu8 content was observed; the highest levels occurred in sacral cord. Dorsal cord content was higher than that of ventral cord at all spinal segments. Immunohistochemical staining supported and refined the latter observation: a dense network of perikarya and fibers was found in Laminae I and II of the dorsal horn. Cell bodies were frequently observed in lamina IV. Additional terminals were seen around the central canal and in the ventral gray matter, often outlining perikarya of motor neurons. Total Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity could be fractionated into two main components using gel filtration chromatography. Nearly half of the total immuno-reactivity eluted as a high molecular weight peptide; the other half which co-eluted with Met5-enkephalin-Arg6-Gly7-Leu8 was further identified to be authentic Met5-enkephalin-Arg6-Gly7-Leu8 on reverse phase high pressure liquid chromatography. The present data, in conjunction with our previous study of Met5-enkephalin and Met5-enkephalin-Arg6-Phe7 indicates that all opioid peptides derived from preproenkephalin A are present in spinal cord and most likely are stored in the same neurons. Immunohistochemical localization of Met5-enkephalin-Arg6-Gly7-Leu8 in dorsal and ventral cord suggest a role for this peptide in both sensory and motor integration.     

Distribution of proenkephalin-derived peptides in the brain of Rana esculenta

The immunocytochemical distribution of authentic proenkephalin-containing perikarya and nerve fibers in the brain of Rana esculenta was determined with antisera directed toward different epitopes of preproenkephalin. The pattern of proenkephalinlike immunoreactivity was similar with antisera directed toward [Met5]-enkephalin, [Met5]-enkephalin-Arg6, [Met5]-enkephalin-Arg6-Phe7, [Leu5]-enkephalin, and metorphamide; however, the intensity of the labelling varied depending on the target antigen. Proenkephalin-containing perikarya were located in all major subdivisions of the brain except the metencephalon. In the telencephalon, immunoreactive perikarya were detected in the dorsal, medial, and lateral pallium; the medial septal nucleus; the dorsal and ventral striatum; and the amygdala. In the diencephalon, immunoreactive perikarya were detected in the preoptic nucleus, in the dorsal and ventral caudal hypothalamus, and in an area that appeared to be homologous to the paraventricular nucleus. In the mesencephalon, numerous immunoreactive perikarya were detected in layer 6 of the optic tectum and a few scattered perikarya were detected in layer 4 of the optic tectum. Immunoreactive perikarya also occurred in the laminar nucleus of the torus semicircularis. In the rhombencephalon, immunoreactive perikarya were detected in the obex region and the nucleus of the solitary tract. Immunoreactive fibers of varying density were observed in all major subdivisions of the brain with the densest accumulations of fibers occurring in the dorsal pallium, the lateral and medial forebrain bundles, the amygdala, the periventricular hypothalamus, the superficial region of the caudolateral brainstem, and in a tract that appeared to be homologous to the tractus solitarius. The extensive system of proenkephalin-containing perikarya and nerve fibers in the brain of an amphibian showed many similarities to the distribution of proenkephalin-containing perikarya and nerve fibers previously described for the amniote brain.     

Analgesic and convulsant effects of guanidinoethylmercaptosuccinic acid (GEMSA)--a potent enkephalin convertase inhibitor

We studied behavioral effects of the intraventricularly and intrathecally administered guanidinoethylmercaptosuccinic acid (GEMSA) - a potent inhibitor of enkephalin convertase. When given intraventricularly in doses of 3 and 6 micrograms, GEMSA elicited analgesia; after doses of 12.5 and 25 micrograms the explosive motor behavior and convulsions occurred. Following the intrathecal administration of GEMSA (12.5, 25 and 50 micrograms), an increase in the tail-flick latency was observed; moreover that drug potentiated analgesic effects of the intrathecally applied Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Gly7-Leu8. All the above effects of GEMSA were significantly attenuated by naloxone. The rats subjected to chronic pain showed a weaker analgesic response to the intrathecally injected GEMSA. The 3H-GEMSA binding to enkephalin convertase in the spinal cord of these rats produced only a slight increase in KD; besides, no changes in the enzyme activity were observed. The study shows that GEMSA has a potent pharmacological action in the central nervous system. Furthermore, this effect is partly due to the influence of GEMSA on endogenous opioid peptide systems, possibly on proenkephalin A.     

Distribution and development of proenkephalin-like immunoreactivity in the Lumbar spinal cord of the chicken

Met5-enkephalin- (Met-ENK), Leu5-enkephalin-, Met5-enkephalin-Arg6-Phe7-, metorphamide- and BAM 22P-like peptides could be detected in the lumbar spinal cord of the chicken by immunocytochemistry and/or high performance liquid chromatography. However, a peptide identical to Met5-enkephalin-Arg6-Gly7-Leu8 could not be detected in the lumbar spinal cord of the chicken using an antiserum that was capable of detecting the octapeptide in mammalian tissues. Nerve fiber- and terminal-like processes containing proenkephalin-derived peptides were concentrated in the superficial laminae of the dorsal horn and along the midline rostral to the central canal. A lesser concentration of processes containing proenkephalin-derived peptides occurred in the medial and lateral motor columns of the ventral horn. The level of total radioimmunoassayable Met-ENK in the lumbar spinal cord of the chicken embryo increased more than 1000-fold between day 4.5 and day 18. A schedule of curare administration that had previously been shown to prevent naturally occurring somatic motoneuron death in the chicken lumbar spinal cord resulted in a two-fold increase in total radioimmunoassayable Met-ENK in the lumbar spinal cord.     

Characterization of metorphamide-like immunoreactivity in the zona incerta and lateral hypothalamus: co-localization with alpha-melanocyte-stimulating hormone-like immunoreactivity

Double-staining in either vibratome or paraffin sections using contrasting chromogens revealed an alpha-melanocyte-stimulating hormone (alpha-MSH)-containing cell group in the arcuate nucleus, a metorphamide-containing cell group in the paraventricular hypothalamus, and an extensive group of magnocellular perikarya in the zona incerta (ZI) and the lateral hypothalamus (LH) that appeared to contain both antigens. Staining of adjacent paraffin sections also suggested that most (and perhaps all) of the magnocellular perikarya in the ZI and LH that contained metorphamide-like immunoreactivity also contained alpha-MSH-like immunoreactivity. Metorphamide-like immunoreactivity in the ZI and the LH was abolished by absorption of the antiserum with metorphamide but was unaffected by absorption with alpha-MSH. alpha-MSH-like immunoreactivity in the ZI and LH was abolished by absorption of the antiserum with alpha-MSH but was unaffected by absorption with metorphamide. Antisera directed against [Met5]-enkephalin (Met-ENK), [Met5]-enkephalin-Arg6,Gly7,Leu8 (ENK-8), [Met5]-enkephalin-Arg6,Phe7 (ENK-7), neuropeptide Y, and FMRF-amide did not stain magnocellular perikarya in the ZI and LH. Pretreatment of paraffin sections with trypsin resulted in the appearance of [Met5]-enkephalin-Arg6-like immunoreactivity in the ZI and LH. Pretreatment of paraffin sections with trypsin did not reveal any occult Met-ENK-, ENK-7- or ENK-8-like immunoreactivity in either the ZI or the LH. These observations indicate that magnocellular neurons in the ZI and LH contain both a metorphamide-like and an alpha-MSH-like peptide but do not express either the preproenkephalin or the prepro-opiomelanocortin48 gene.     

Endogenous opioid systems regulate growth of neural tumor cells in culture

Endogenous opioid systems (i.e., opioids and opioid receptors) play a role in neural cancer. Using a tissue culture system of S20Y murine neuroblastoma to assess the effects of opioids on growth, [Met5]-enkephalin was the most potent compound to influence cell replication. With a median effective concentration of 10(-10) M, this peptide inhibited cell proliferation in a stereospecific and naloxone-reversible manner. [Met5]-Enkephalin depressed both DNA synthesis and mitosis. [Met5]-Enkephalin was detected in neuroblastoma cells by radioimmunoassay, and was found to increase in concentration in culture media over time, suggesting that these cells produced the peptide. Immunocytochemistry showed [Met5]-enkephalin-like activity in the cortical cytoplasm, but not the cell nucleus, of neuroblastoma cells. Binding of [3H]-[Met5]-enkephalin specific and saturable, and Scatchard analysis yielded a Kd of 1.2 +/- 0.1 nM and a binding capacity of 50.2 +/- 4.3 fmol/mg protein. [Met5]-Enkephalin also depressed the growth of N115 murine neuroblastoma, SK-N-MC human neuroblastoma, and HT-1080 human fibrosarcoma. These results indicate that [Met5]-enkephalin, a naturally occurring pentapeptide that is derived from proenkephalin A, is a potent inhibitor of cell growth. Since cancer cells produce [Met5]-enkephalin, and contain a binding site to this ligand, endogenous opioid systems appear to control cell proliferation by an autocrine mechanism.     

Presence of Met-enkephalin-Arg6-Phe7 in molluscan neural tissues

An extract of pedal ganglia of Mytilus edulis was fractioned by high-pressure liquid chromatography by use of a reverse-phase column. Peak fractions with the same retention time as Met-enkephalin-Arg6-Phe7 were subjected to binding assays in invertebrate neural tissue. The results showed that these fractions have the same binding activities as authentic Met-enkephalin-Arg6-Phe7. The heptapeptide from these fractions was purified by high-pressure liquid chromatography under isocratic conditions. Sequential amino acid analysis showed this peptide to have the same primary structure as Met-enkephalin-Arg6-Phe7. These results indicate that invertebrates such as Mytilus edulis possess enkephalinergic systems similar to those found in in higher organisms.     

Changes in dynorphin, enkephalin and cholecystokinin content of hippocampus and substantia nigra after amygdala kindling

Amygdaloid kindling of rats produced an increase in hippocampal Met5-enkephalin-Arg6-Gly7-Leu8 and cholecystokinin immunoreactivities and simultaneously a decrease in dynorphin A1-8 content. In substantia nigra Met5-enkephalin-Arg6-Gly7-Leu8 was increased and no change was observed in dynorphin A1-8 content. These data suggest that specific alterations of neuropeptides in limbic and extrapyramidal circuits are prominent manifestations of the kindling process or kindled seizures.     

Angiotensin-converting enzyme associated with Torpedo california electric organ membranes

Torpedo electric organ contains high concentrations of angiotensin converting enzyme (ACE) like activity, cleaving [Leu5]enkephalin at the Gly3-Phe4 peptide bond. Most of the activity cosediments with the cell membranes. The enzymatic preparation from membranes is inhibited by low concentrations of the ACE inhibitors, SQ 14225 and SQ 20881 (IC50 of 0.6 and 15 nM, respectively), and is weakly inhibited by the neutral endopeptidase inhibitors, phosphoramidon and thiorphan (IC50 of 30 microM and ca. 70 nM, respectively). The enzyme degrades hippuryl-His-Leu and is activated by NaCl. Hippuryl-His-Leu and [Leu5]enkephalin are degraded with Km of 93 and 41 microM, and Vmax of 21 and 10 nmol/mg protein/min, respectively. The specific activity of the ACE-like activity in homogenates of Torpedo electric organ is relatively high (6.3 nmol hippuryl-His-Leu/mg protein/min); this value is similar to that obtained for rat lung and rat striatum.     

Autoimmunoregulation: differential modulation of CD10/neutral endopeptidase 24.11 by tumor necrosis factor and neuropeptides.

Neutral endopeptidase (NEP) 24.11 appears to be an important enzyme in both vertebrate and invertebrate autoimmunoregulation. Activation of human or invertebrate immunocytes that express NEP with substrates such as monokines and neuropeptides results in its increased expression, in other words, upregulation. However, since certain neuropeptides are also substrates for NEP, these activated immunocytes will respond to neuropeptides only at higher concentrations, thus downregulating the response. Specifically, in tumor necrosis factor (TNF)-treated immunocytes, we demonstrate the effects of increased NEP expression on altering the stimulatory activities of the neuropeptides met-enkephalin, melanocyte-stimulating hormone and substance P. We demonstrate the significance of NEP in modulating these responses through the use of specific enzyme inhibitors such as phosphoramidon, thiorphan and captopril. Furthermore, we present evidence suggesting that the individual variations seen in immunocytes from both different and the same donors to activating substances may reflect fluctuating levels of NEP expressed in response to endogenous stimuli. These results indicate that NEP is a highly significant factor in controlling the response(s) of certain immunocytes in man and higher invertebrates to the influence of biologically active substances such as monokines and neuropeptides.     

HIV gp120 alteration of DAMA and IL-1α induced chemotaxic responses in human and invertebrate immunocytes

The effects of a synthetic peptide fragment of human immunodeficiency virus gp120 (HIV gp120) on opioid (D-ala²-D-met⁵ enkephalinamide; DAMA) and interleukin-1 (IL-1) induced chemotactic responses in human granulocytes and monocytes and invertebrate (Mytilus edulis) immunocytes were studied. Both DAMA and IL-1 increased the velocity of cell migration from both species and the response is chemotactic (e.g. directed). Non-treated control cells move randomly or not at all. The addition of gp120 to DAMA or IL-1 treated human granulocytes or monocytes results in a slower movement which is chemokinetic (loss of directionality or random) in nature. A similar phenomenon occurs in the invertebrate immunocytes. If gp120 alone is added, it inhibits the movement of spontaneously active human granulocytes and Mytilus edulis immunocytes. In contrast, it stimulates chemokinesis of spontaneously active human monocytes. These responses occur immediately after addition of the peptide. Based on experiments with the selective calcium channel antagonist nimodipine, it appears that the gp120 causes its effects by irreversible binding to a calcium channel. Our results suggest a universal inhibitory mechanism is occuring since the invertebrate immunocytes must recognize HIV gp120 peptide to result in this effect, possibly through a CD4 or other type of surface determinant.     

Carboxypeptidase B-like activity for the processing of enkephalin precursors in the membrane component of bovine adrenomedullary chromaffin granules

Trypsin and carboxypeptidase B-like (CPB-like) peptidases should be involved in processing proenkephalin to form the small biologically active enkephalins. A carboxypeptidase B-like activity from the soluble fraction of bovine adrenomedullary chromaffin granules which converts 125I-(Met)-enkephalin-Arg6 to 125I-(Met)enkephalin has previously been described and characterized (1,2). In this study, CPB-like activity in the membrane bound component of chromaffin granules is characterized and compared with that in the soluble fraction. Membrane and soluble CPB activities cleaved 125I-(Met)enkephalin-Arg6 or 125I-(Met)enkephalin-Lys6 to form 125I-(Met)enkephalin. Like the soluble enzyme, the CPB-like activity in the membrane component had a pH optimum of 6.0, was inhibited by thiol agents (PCMPSA, CuCl2) and metal ion chelators (EDTA, 1,10-phenanthroline), and was stimulated by Co++. The membrane CPB-like activity appeared to be an intrinsic membrane protein, since 80% of the activity remained with the membranes after washing with 1.0 M NaCl. Membrane and soluble CPB-like activities in chromaffin granules appear to be similar enzymes.     

Cloning and characterization of Aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in Aplysia californica

Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.     

Endogenous opioids regulate cell proliferation in the retina of developing rat

The role of endogenous opioids and opioid receptors (endogenous opioid systems) in modulating cell proliferation in the developing mammalian retina was examined in 1-day-old rats. In contrast to a labeling index (LI) of 35.8% in control animals, administration of the opioid peptide [Met5]-enkephalin (100 micrograms/kg) significantly reduced (10.6%) the proportion of cells incorporating [3H]thymidine; concomitant injection of 1 mg/kg naloxone blocked the inhibitory effects of [Met5]-enkephalin on cell division. Naloxone (1 mg/kg) alone did not alter the LI. The interruption of endogenous opioid-opioid receptor interaction by naltrexone (50 mg/kg), a potent opioid antagonist, was accompanied by a significant increase (6.4%) in the LI relative to control levels. Immunocytochemical experiments revealed the presence of enkephalin-like immunoreactivity, with staining of the cortical cytoplasm of proliferating and differentiating retinal cells recorded; no immunoreactivity was noted in the adult retina. In vitro autoradiography using 125I-[Met5]-enkephalin indicated that [Met5]-enkephalin binding sites were localized to the developing retina; no binding of the radiolabeled ligand was recorded in the adult retina. These results demonstrate the presence of growth-related endogenous opioids and opioid receptors in the developing mammalian retina, but not in adult retina, and suggest that endogenous opioids serve as natural inhibitory trophic factors that tonically regulate cell proliferation.     

Proenkephalin-A derived peptides do not modulate cardiovascular effects of epinephrine on the isolated rat atrial preparations

The catecholamines and the opioid peptides are found to be co-localized in the adrenomedullary chromaffin cells. They are co-secreted from the chromaffin granules in response to various stimuli. The stress-induced released of epinephrine is known to exert its effect on the cardiovascular system resulting in the changes in heart rate and blood pressure. However, the role of the co-released proenkephalin-A derived peptides has not been extensively characterized. Previous work from several investigators suggested that the peptides modulate cardiac functions of the catecholamines. There is considerable conflicting results among these reports. Results from the isolated rat atrial preparation indicated that enkephalins attenuated the increase in atrial rate induced by norepinephrine through restriction of the calcium fluxes. Nonetheless, others reported insensitivity of the enkephalins in similar or different test systems. We further re-examined these discrepancies using the isolated rat atrial preparation to investigate the opioid peptide modulatory effect on the cardiovascular changes induced by exogenous epinephrine. Alterations in rate and force of contraction resulting from epinephrine and the peptides were both studied in parallel. The opioid peptides used in this study were [Met5]-enkephalin (ME), [Leu5]-enkephalin (LE), FMRFamide, [Met5]-enkephalyl-Arg6-Phe7 (MEAP), peptide E, and the non-selective opioid agonist, etorphine. We report here that none of the opioid peptides were effective in alleviating or attenuating the increase in heart rate and developed tension caused by epinephrine. The peptides did not affect the basal beating rate nor the force of contraction. Thus, the present results clearly demonstrate the insensitivity of the enkephalins in modulating the cardiac effects of epinephrine. They further indirectly support the prejunctional synaptic nerve endings as the potential peripheral site of action of the peptides.     

Possible role of increased brain methylation in methionine sulfoximine epileptogenesis: effects of administration of adenosine and homocysteine thiolactone

An intraventricular pulse of [14COOH]L-methionine to mice pretreated with the convulsant L-methionine-dl-sulfoximine (MSO) resulted in significantly higher than control specific radioactivity values of cerebral [14COOH]L-methionine (Met), [14COOH]S-adenosyl-L-methionine (AdoMet) and [14COOH]S-adenosyl-L-homocysteine (AdoHcy). MSO administration (3 hr) also decreased brain steady-state levels of Met, AdoMet, and AdoHcy. Following an intraventricular pulse of [3H-methyl]L-methionine, the levels of [3H-methyl]phosphatidylmonomethylethanolamine and of membrane associated and soluble [3H-methyl]carboxylmethylated proteins were increased over corresponding saline-treated controls. The activity of cerebral histamine N-methyltransferase was also increased after MSO treatment. The administration of a combination of adenosine and homocysteine thiolactone to MSO-pretreated animals counteracted the MSO-induced decreases in brain Met, AdoMet, and AdoHcy as well as the increase in histamine N-methyltransferase activity. In addition, administration of adenosine together with homocysteine thiolactone decreased the incidence of, and increased the latency to MSO seizures, with the most effective anticonvulsant action occurring when cerebral AdoHcy levels were at their highest.     

A neuroimmunoregulatory-like mechanism responding to stress in the marine bivalve Mytilus edulis

Mytilus edulis has been the subject of recent studies to determine whether the relationship between the immune and nervous systems seen in vertebrates also exists in invertebrates. In the present study the effects of experimentally induced "stressful" stimuli on immunoactive hemocytes were studied in this mollusc. This subpopulation of invertebrate blood cells, resembling vertebrate granulocytes, has been previously shown to produce and react to opioid peptides. Their activation, like that of vertebrate immunocytes, expresses itself in distinctive conformational changes preceding cellular mobilization. The cellular response to "stress" observed is the same as that to the administration of exogenous mammalian opioid peptides. This strongly suggests that under the conditions of stressful stimuli, the immune/defense system can be altered by endogenous neuropeptides. The involvement of opioids in neuroimmunoregulatory phenomena appears to have a long evolutionary history.     

Absence of NK1 receptors in human blood lymphocytes and granulocytes

Substance P is a peptide that exerts its activity through the interaction with specific receptors that are distributed in different brain areas. Given the potential of NK1 receptor antagonists as antidepressants, the availability of a peripheral model of NK1 receptors would be particularly relevant for the possibility to perform studies in samples of patients. Therefore, with the present study we aimed to explore the possible existence of NK1 receptors by means of [3H]SR140333 and [125I]BHSP that behave as, respectively, antagonist and agonist, at this level, in human blood lymphocytes and granulocytes of healthy donors. The results of the present study failed to detect the presence of a high-affinity and saturable binding of [3H]SR140333 and [125I]BHSP in human blood cells, whereas a specific binding for both compounds was found in rat cerebral cortex that was used as the control tissue. These findings would question the presence of NK1 receptors in human circulating cells.