Immunohistochemistry of opioid peptides in the guinea pig endocrine pancreas

Summary Previous immunochemical investigations have demonstrated various opioid peptides in the pancreas. However, controversies exist related to the cellular localization of these peptides in the endocrine pancreas. Therefore, the guinea pig endocrine pancreas was immunohistochemically investigated for the presence of opioid peptides derived from pro-dynorphin, pro-enkephalin or pro-opiomelanocortin. Immunoreactivities were demonstrated on serial semithin sections by the peroxidase anti-peroxidase technique. In routinely immunostained sections, immunoreactivities for dynorphin A and -neo-endorphin were localized in pancreatic enterochromaffin cells, but not in islet cells. Immunoreactivity for Met-enkephalin was confined exclusively to B-cells and was localized only in some secretory granules. However, pre-treatment of semi-thin sections with trypsin and carboxypeptidase B led to a marked increase of Met-enkephalin immunoreactivity in B-cells. In addition, immunoreactivities for Met-enkephalin-Arg-Gly-Leu and bovine adrenal medulla dodecapeptide could be demonstrated in B-and A-cells, and -endorphin immunoreactivity was localized in A-cells. In no case, however, were immunoreactivities detected for bovine adrenal medulla docosapeptide, peptide F, corticotropin, melanotropin or dynorphin 1–32. The immunohistochemical findings indicate that opioids of different peptide families are present in the guinea pig endocrine pancreas. Since several opioid peptides of the corresponding pro-hormones could be demonstrated in the reference organs but not in the pancreas, it is concluded that the biosynthetic pathways of the respective precursors are different from those in the adrenal medulla or in the pituitary.     

Biochemical evidence of specific trypsin-chymotrypsin inhibitors in the rhynchobdellid leech, Theromyzon tessulatum

The presence of two specific trypsin-chymotrypsin inhibitors from head parts of the rhynchobdellid leech Theromyzon tessulatum is reported. Two proteins, anti-trypsin chymotrypsin A (ATCA; 14636.6 +/- 131 Da) and anti-trypsin-chymotrypsin B (ATCB; 14368 +/- 95 Da) were purified by size exclusion and anion-exchange chromatography followed by reversed-phase HPLC. Based on amino-acid composition, N-terminal sequence determination (MELCELGQSCSRD-NPQPSNM), matrix assisted laser desorption-time of flight measurement (MALDI-TOF), trypsin mapping comparison, inhibition constant determination (Ki), and influence on amidolytic activity of different serine proteases, it is demonstrated that ATCA and ATCB are novel and highly potent serine-protease inhibitors of trypsin and chymotrypsin (ATCA: 350fM towards trypsin and chymotrypsin; ATCB: 400 and 75 fM towards trypsin and chymotrypsin, respectively). It is further surmised that ATCA and ATCB are linked, in that ATCB would lead to the formation of ATCA after loss of few amino acid residues.     

Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.     

Non-opioid actions of opioid peptides

Beside the well known actions of opioid peptides on mu-, delta- and kappa-opioid receptors, increasing amount of pharmacological and biochemical evidence has recently been published about non-opioid actions of various opioid peptides. These effects are not abolished by naloxone treatments. Such non-opioid effects are observed both in nervous tissues and in the cellular elements of the immune system. Peptides exhibiting non-opioid effects include beta-endorphin, dynorphin A, nociceptin/OFQ, endomorphins, hemorphins and a number of Proenkephalin A derived peptides, such as Met-enkephalin, Met-enkephalin-Arg-Phe (MERF) and bovine adrenal medullary peptide (BAM22). Non-opioid actions are exerted through different neuronal receptors, e.g., dynorphin hyperalgesia through NMDA receptor, Met-enkephalin induced regulation of cell growth through zeta receptors, pain modulation by nociceptin through ORL-1 or NOP receptors, while BAM22 acts through sensory neuron specific G protein-coupled receptors (SNSR). We have investigated Met-enkephalin-Arg-Phe (MERF) and its analogues by the means of direct and indirect radioligand binding assays. It has been found that in addition to kappa(2) and delta-opioid receptors, MERF can act also through sigma(2)- or probably via FMRF-NH(2) receptors in rat cerebellum. A role of functionally assembling heterodimer receptors in mediating the non-conventional actions of these peptide ligands can not be excluded as well.     

Effects of exercise training on brain opioid peptides and serum LH in female rats

In order to investigate the effects of long-term exercise training on brain endorphin systems, and the latter's possible effects on the hypothalamic-pituitary-gonadal axis, female Wistar rats were subjected to daily treadmill running. A sedentary control group was also employed. After 8 weeks of training, and just prior to sacrifice, one-half of each group received a final fatiguing bout of exercise. Thus the final four groups consisted of a trained-fatigued (TF), trained-nonfatigued (TN), control-fatigued (CF), and control-nonfatigued (CN) group. Regional brain levels of beta-endorphin (beta E), methionine enkephalin and leucine enkephalin (LE) were assayed with independent RIAs from the nucleus accumbens, cortex, caudate-putamen, septum, amygdala, anterior and posterior hypothalamus, substantia nigra and ventral tegmentum. Diestrus serum levels of luteinizing hormone (LH), follicle stimulating hormone and prolactin (PRL) were also determined. Fatiguing resulted in a decrease in serum LH levels as well as an increase in beta E content in the nucleus accumbens, and LE content in the ventral tegmentum. Finally, TF animals exhibited less LE in the amygdala than the TN rats. Taken together, these changes in brain endorphins may indicate an acute, fatigue-running modulation of the hypothalamic-pituitary-gonadal axis.     

Characterization of immunoreactive neurons in the central nervous system of the leech Theromyzon tessulatum using monoclonal antibodies

Two mouse hybridomas producing monoclonal antibodies Tt9 and Tt 159 directed against antigens of supraesophageal ganglia of the leech T. tessulatum were selected to study the neuroendocrine control of osmoregulation in this species. One, Tt 159 reacted with an antigenic determinant of cells recognized by an anti-angiotensin antibody, the other, Tt 9, with neurons immunoreactive to the anti-vasopressin.     

Immunohistochemical localization of polypeptide hormones in endocrine cells of the digestive tract of Branchiostoma lanceolatum

Summary The digestive tract of the cephalochordate Branchiostoma lanceolatum was investigated with regard to occurrence and distribution of endocrine cells. By the use of the peroxidase-antiperoxidase (PAP) technique, cells in the gut epithelium reacting with antisera against 8 different mammalian polypeptide hormones were localized. Positive reactions were obtained with antisera against the four mammalian islet hormones (insulin, glucagon, pancreatic polypeptide, somatostatin) and against secretin, vasoactive intestinal polypeptide, pentagastrin and neurotensin. No immunoreactivity was found with antisera against members of the lipotropin family (ACTH, met-enkephalin, -endorphin), against big-gastrin, cholecystokinin, substance P and moulin. The exact mapping of the different polypeptide immunoreactive cells throughout the digestive tract of Branchiostoma lanceolatum is presented.     

Immunohistochemical localisation of natriuretic peptides in the heart and brain of the gulf toadfish Opsanus beta

Summary The distribution of natriuretic peptide immunoreactivity was determined in the heart and brain of the gulf toadfish Opsanus beta using the avidin-biotin peroxidase technique. Four antisera were used: the first raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); the third raised against rat atrial natriuretic peptide; and the fourth raised against eel atrial natriuretic peptide. Natriuretic peptide- and porcine brain natriuretic peptide-like immunoreactivity was observed in all cardiac muscle cells of the atrium. In the ventricle, natriuretic peptide-like immunoreactivity was found in all cardiac muscle cells, however porcine brain natriuretic peptidelike immunoreactivity was confined to muscle cells adjacent to the epicardium. There was no discernible difference in the distribution of natriuretic peptide-like immunoreactivity and porcine brain natriuretic peptide-like immunoreactivity in the brain. Immunoreactive perikarya were observed only in the preoptic region of the diencephalon, and many immunoreactive fibres were found in the telencephalon, preoptic area, and rostral hypothalamus, lateral to the thalamic region. There was no immunoreactivity in any region of the hypophysis. A pair of distinct immunoreactive fibre tracts ran caudally from the preoptic area to the thalamic region, from which fibres extended to the posterior commissure, area praetectalis, dorsolateral regions of the midbrain tegmentum, and tectum. Many immunoreactive fibres were present in the rostral regions of the inferior lobes of the hypothalamus and in the dorsolateral and ventrolateral aspects of the rhombencephalon. No immunoreactivity was observed in the heart and brain using rat atrial natriuretic and eel natriuretic peptide antisera. Although the chemical structure of natriuretic peptides in the heart and brain of toadfish is unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic and/or porcine C-type natriuretic peptides. The presence of natriuretic peptides in the brain suggests that they could be important neuromodulators and/or neurotransmitters.     

Characterization of opioid peptides from maternal and fetal sheep adrenal glands

Enkephalin immunoreactive material from adrenal glands was characterized both in maternal and fetal sheep at various gestational ages. Whole gland extracts from both maternal and fetal sheep contained three major peaks of Enk immunoreactivity corresponding to apparent molecular weights of 10,000, 2800, and less than 1200 daltons. The majority of maternal adrenal Enk immunoreactivity was found in medullary tissue, although cortex also contained low but detectable amounts. This was also the case in newborn lambs and 139 day fetuses, where adrenal cortex was sufficiently developed to allow extraction and quantitation of opioid material. In fetuses at mid-gestation (70-80 days), adrenal medullary Enk immunoreactivity was approximately 75% of maternal values. Met-Enk and Leu-Enk content in 139 day fetal medulla were 70 and 76% of maternal values respectively, while newborn Met- and Leu-Enk medullary content were similar to maternal values. The molar ratio of Met-Enk to Leu-Enk was approximately 4:1 in both maternal and fetal adrenal medulla, and 2:1 in adrenal cortex, suggesting different synthetic processing of opioid peptides in the two tissues. The early appearance of significant levels of adrenal medullary Enk immunoreactivity and subsequent development paralleling that of catecholamines suggest a predominant role for adrenal enkephalins in regulation of fetal cardiovascular function early in gestation.     

Immunohistochemical localization of chromogranin A and B in endocrine cells of the alimentary tract of the adult lizard Podarcis sicula

The distribution, argyrophilia, and the possible amine/peptide co-localizations in endocrine cells immunoreactive (IR) to antisera against chromogranin A (CgA) and chromogranin B (CgB) in the alimentary tract of the lizard Podarcis sicula have been investigated using novel monoclonal antibodies. Many CgA-IR and CgB-IR cells were found in the tract, except in the distal small intestine. Almost all chromogranin-IR cells (Cgs-IR) were also argyrophilic with parallel intensity. Some CgA-IR and CgB-IR cells did not display co-localized amines or peptides. CgA or CgB or both were found co-localized, with some local differences, in almost all serotonin-IR, histamine-IR, substance P-IR and gastric peptide tyrosine tyrosine (PYY)-IR cells. Moreover, both Cgs were co-localized only in some somatostatin-IR cells, whereas neurotensin-IR, gastrin/cholecystokinin-IR, pancreatic polypeptide-IR and intestinal PYY-IR cells did not show any co-localization with Cgs. The presence of Cgs in the endocrine cells was heterogeneous with regard to the complex interrelationship with their amine/peptide content. Consequently, Cgs cannot be considered as universal markers of all endocrine cell types.     

Immunohistochemical localization of neuropeptide Y-like substance in the brain and hypophysis of the brown hagfish,Paramyxine atami

The distribution of neuropeptide Y-like immunoreactivity in the brain and hypophysis of the brown hagfish, Paramyxine atami, was examined by use of the peroxidase-antiperoxidase method. Immunoreactive cells were found in two areas of the brain, the nucleus hypothalamicus of the diencephalon and the ventrolateral area of the caudal tegmentum, at the level of the nucleus motorius V–VII. The labeled cells of the nucleus hypothalamicus were loosely grouped and recognized as bipolar neurons. Immunolabeled fibers were widely distributed in the brain, showing the highest density in the diencephalon. They were sparse, or absent, in the olfactory bulb, habenula, primordium hippocampi, neurohypophysis, corpus interpedunculare, and dorsolateral area of the medulla oblongata. The fibers appeared to project exclusively from the ventral hypothalamus to various other portions of the brain: the anterolateral areas of the telencephalon via the basal hypothalamus, the pars dorsalis thalami, the dorsocaudal region of the mesencephalon, and the ventromedial portions of the tegmentum and anterior medulla oblongata. These findings suggest that, in the brown hagfish, NPY-like substance is involved in neuroregulation of various cerebral areas, but it may be of little significance in the control of pituitary function.     

Two-dimensional ultrastructural elements lead to three-dimensional reconstruction of protuberances on the cocoon membrane of the leech, Theromyzon tessulatum

Protuberances on the cocoon surface of the leech, Theromyzon tessulatum, are roughly parallel rows of triangular prisms arranged equidistantly to each other on the outer surface of the cocoon membrane. The distance between neighboring protuberances is approximately 1.6 microm, the height approximately 0.5 microm and the semi-width approximately 0.3 microm. The fibrillar arrangement within the protuberance maintains some elements of the helicoids found within the cocoon membrane but a high proportion of large holes disrupt the symmetry of the protuberance ultrastructure. A procedure for 3D reconstruction of the protuberance using the complementarity between the paratangential and normal sections through the cocoon is presented. Our results demonstrate that the ultrastructure of protuberances show elements of a twisted fibrillar arrangement, but the demands of filling a narrow space ruled by acute angles appears to cause a high degree of ultrastructural disorganization.     

Life cycle and habitat ofGlossiphonia paludosa (Hirudinea: Glossiphoniidae), a new leech for the Netherlands

The widely distributed but rare leechGlossiphonia paludosa (Carena) was collected for the first time in The Netherlands in 1988 in a eutrophic ditch in the nature reserve ‘De Regulieren’ near Geldermalsen. The habitat is described briefly and, for the first time, the life cycle ofG. paludosa is presented.     

Cell dynamics during cocoon secretion in the aquatic leech, Theromyzon tessulatum (Annelida: Clitellata: Glossiphoniidae)

One distinguishing feature of clitellate annelids is the presence of specialized segments comprising the clitellum, whose primary function is to secrete a cocoon. Using histological analyses, we have documented cell types (I-V) and cellular processes associated with cocoon secretion in the aquatic leech, Theromyzon tessulatum. Our data indicate that the bulk of the cocoon's biomass arises from precursor cells of a single type that hypertrophy and proliferate ∼1 week prior to egg laying, and then differentiate into either of two cell types (i.e., Type II or Type III) depending on their position within the clitellum. Type II cells are concentrated along the lateral edges and venter of the clitellum and secrete alcian blue-staining granules that form opercula (i.e., glue-like material that seals both cocoon ends), while Type III cells populate the dorsal midline and secrete azocarmine-staining granules that build the cocoon wall. Both cell types occupy spaces between deep muscle layers and extend long-neck tubules to the surface epithelium as they fill with granules a few days prior to egg laying. Other cell types appear to make minor contributions to the cocoon (e.g., Type I, Type IV) or have supporting or signaling roles (e.g., Type V). Our observations suggest that post-translational modification (i.e., glycosylation) of the same core protein(s) distinguishes the granules of Type II/III cells, and that the default state of the Type II/III precursor may be evolutionarily linked to secretory cells in basal polychaetes.     

Immunohistochemical localization of chromogranin A and B in the endocrine cells of the alimentary tract of the green frog,Rana esculenta

Novel monoclonal antibodies to human chromogranin A (CgA) and chromogranin B (CgB) were used to investigate the presence of immunoreactive (-IR) elements in the alimentary tract of the green frog Rana esculenta. Numerous CgA-IR and a few CgB-IR endocrine cells were found within the gut mucosa, from the oesophagus to the cloaca, with some local differences in density. Co-localization studies demonstrated that they were costored in almost all the serotonin-IR, the amylin-IR or islet amyloid polypeptide-IR cells and in the peptide tyrosine tyrosine-IR cells located proximal to the pylorus, but not in those located in more caudal tracts. No other co-localization was demonstrated; substances investigated included somatostatin, substance P, gastrin/cholecystokin, glucagon, glycentin, bombesin, secretin and neurotensin. CgA-IR and CgB-IR cells nearly always displayed argyrophilia with the Grimelius silver method     

Apparent involvement of opioid peptides in stress-induced enhancement of tumor growth

Exposure to stress has been associated with alterations in both immune function and tumor development in man and laboratory animals. In the present study, we investigated the effect of a particular type of inescapable footshock stress, known to cause an opioid mediated form of analgesia, on survival time of female Fischer 344 rats injected with a mammary ascites tumor. Rats subjected to inescapable footshock manifested an enhanced tumor growth indicated by a decreased survival time and decreased percent survival. This tumor enhancing effect of stress was prevented by the opiate antagonist, naltrexone, suggesting a role for endogenous opioid peptides in this process. In the absence of stress, naltrexone did not affect tumor growth.     

Interaction of opioid peptides and other drugs with multiple δncx binding sites in rat brain: Further evidence for heterogeneity

Recent pharmacological data strongly support the hypothesis of δ receptor subtypes as mediators of both supraspinal and spinal antinociception (δ₁ and δ₂ receptors). In vitro ligand binding data, which are fully supportive of the in vivo data, are still lacking. A previous study indicated that [³H][ -Ala², -Leu⁵]enkephalin labels two binding sites in membranes depleted of μ binding sites by pretreatment with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole-HCI (BIT). The main goal of the present study was to develop a ligand-selectivity profile of the two δ_ncx binding sites. The data indicated that naltrindole and oxymorphindole were relatively selective for site 1 (20-fold). [ -Ser²,Thr⁶]Enkephalin and deltorphin-II were only 2.7-fold and 2.2-fold selective for site 1. [ -Pen², -Pen⁵]Enkephalin and deltorphin-I were 80-fold and 38-fold selective for site 2.3-Iodo-Tyr- -Ala-Gly-Phe- -Leu was 52-fold selective for site 1. Morphine had moderate affinity for site 1 (K_i = 16 nM), and was about 11-fold selective for site 1. Thus, of the 10 drugs studied, only DPDPE and DELT-I were selective for site 2. Viewed collectively with other data, it is likely that the δ₁ receptor and the δ_ncx binding site are synonymous.     

The life history,diet and migration of a lake-dwelling population of the leech Alboglossiphonia heteroclita (L.)

Alboglossiphonia heteroclita has an annual life cycle in Tabley Mere, England. Egg-carrying leeches occurred from early June to mid-July. Young were released from parents in August, and the old cohort died soon after. The mean weight of recruited young increased in early autumn, declined over winter due to low feeding activity, and increased again from early spring until the leeches reproduced.The disappearance of leeches from the littoral zone in winter was due to their migration into deeper waters.Six species of snails collected from shallow and deep waters did not harbour leeches inside their shells or mantle cavities at any time of the year. Laboratory experiments confirmed that the leech does not use snails as a refuge.A serological technique identified the gut contents of field-collected A. heteroclita. Leeches had fed most extensively on oligochaetes followed by snails and then chironomids, lightly on Asellus and caddisflies, and scarcely on amphiphods and cladocerans. In laboratory experiments, in which four prey taxa were offered simultaneously to leeches, the descending order of utilization was oligochaetes, chironomids, Asellus and snails.     

Interaction of opioid peptides and other drugs with multiple delta ncx binding sites in rat brain: further evidence for heterogeneity.

Recent pharmacological data strongly support the hypothesis of δ receptor subtypes as mediators of both supraspinal and spinal antinociception (δ₁ and δ₂ receptors). In vitro ligand binding data, which are fully supportive of the in vivo data, are still lacking. A previous study indicated that [³H][ -Ala², -Leu⁵]enkephalin labels two binding sites in membranes depleted of μ binding sites by pretreatment with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole-HCI (BIT). The main goal of the present study was to develop a ligand-selectivity profile of the two δ_ncx binding sites. The data indicated that naltrindole and oxymorphindole were relatively selective for site 1 (20-fold). [ -Ser²,Thr⁶]Enkephalin and deltorphin-II were only 2.7-fold and 2.2-fold selective for site 1. [ -Pen², -Pen⁵]Enkephalin and deltorphin-I were 80-fold and 38-fold selective for site 2.3-Iodo-Tyr- -Ala-Gly-Phe- -Leu was 52-fold selective for site 1. Morphine had moderate affinity for site 1 (K_i = 16 nM), and was about 11-fold selective for site 1. Thus, of the 10 drugs studied, only DPDPE and DELT-I were selective for site 2. Viewed collectively with other data, it is likely that the δ₁ receptor and the δ_ncx binding site are synonymous.     

Heligmosomoides polygyrus: opioid peptides are involved in immune regulation of the histotropic phase of infection

We investigated the potential effect of agonists of opioid receptors in the experimental model of Heligmosomoides polygyrus primary infection. BALB/c mice infected with H. polygyrus were treated with naltrexone, a non-specific antagonist of all three types of opioid receptors (mu, delta, kappa) prior to and during infection. The blockade of opioid receptors affected the pattern and level of immune response induced by H. polygyrus in the histotropic phase of infection, which suggests that an opioid receptor-linked mechanism is involved in the immune response of mice during this phase of infection. Down-regulation of the inflammatory response against fourth-stage larvae appeared to be by endogenous opioids influenced cytokines and nitric oxide production by macrophages in the peritoneum and in the gut as well as migration of leukocytes towards the antigens. Down-regulation of these mechanisms by opioid receptor agonists in vivo might account for the decreased resistance to H. polygyrus infection.