Double immunogold staining method for the simultaneous ultrastructural localization of regulatory peptides

Recent studies have suggested that the morphological characteristics of secretory granules contained within endocrine cells and nerves may be determined largely by their chemical composition. The use of the immunogold staining (IGS) method, which is based on the adsorption of colloidal gold to immunoglobulins, has been used in our laboratory to demonstrate a wide range of intracellular antigens at both the light and electron microscope levels. In this study we have applied a modification of the IGS method for the simultaneous detection of two separate antigens in a single tissue section, using a variety of region-specific antisera to different peptides. Peptide antisera were raised in rabbits or in guinea pigs and these were applied simultaneously or sequentially to grid-mounted ultrathin tissue sections. Antigenic sites were visualized at the electron microscope level using antisera raised in goats, adsorbed to gold particles of 12, 20, or 40 nm. Using this technique we have attempted to investigate the coexistence of multiple antigens in single tissue sections, in particular in single granules; the topographic distribution of molecular forms within one single granule or granule population; the heterogeneity of peptidergic neurons and also the heterogeneity of peptide content in morphologically similar granules. The double immunogold staining procedures described here have proved to be extremely effective for the simultaneous ultrastructural localization of two antigens (peptide-peptide; peptide-propeptide) on a single tissue section. The further development of this technique may provide useful information on neuroendocrine cell dynamics in normal and diseased states.     

Immunoreactivities of gastrin (G-) cells. I. dilution-dependent staining of G-cells by antisera and non-immune sera

Results of immunocytochemical studies reported by several laboratories suggest that gastrin (G-) cells of the stomach show immunoreactivities for various pituitary hormones (ACTH, met-enkephalin, beta-endorphin and growth hormone) in addition to gastrin. By reinvestigating the immunocytochemistry of G-cells we found that these cells exhibited reactivities towards a variety of antisera against enteric, pancreatic and hypophyseal hormones. Gastrin cells can also be "immunostained" by antisera towards proteins unrelated to any peptide hormones (e.g. alpha-fetoprotein antiserum) and by nonimmune sera. Thus the specificity of immunocytochemical findings in G-cells seems to be uncertain. According to our findings the polyvalent immunoreactivities of G-cells may be caused by a distinct binding capacity for IgG molecules. This binding of IgG to G-cells seems to be mediated by the Fab fragments of the IgG molecules which may behave like a basic dye and therefore "immunostain" anionic components within G-cells. Thus the significance of the immunocytochemical proof of peptide hormones within G-cells is limited unless extended specificity controls have been performed. The results of specificity controls performed in this study (adsorption controls, use of ascending dilutions of the primary and secondary antisera, comparison of crude antisera and affinity chromatographically purified antibodies) suggest that corticotropin-lipotropin related peptides are not contained in G-cells.     

Recent development in hormone research

A classical distinction between endocrine cells and neurons cannot be accepted without exception. This dichotomy was first challenged by the concept of neurosecretion. Recent observations indicate that hormone synthesis takes place in many extraendocrine tissues since the gene expression for prohormone synthesis seems to be common for all eukaryotes although the secretion of biological active hormone products is limited by posttranslational processing for differentiated cells. Increasing number of data support the view that regulation of pituitary hormone secretion is under multifactorial control in addition to specific signaling molecular effects of hormone-releasing hormones. Such modulators are co-secreted messengers from hypothalamic sources or co-functioning at the pituitary cell level. Multichannel regulation of pituitary tropic hormones appears to be important for understanding the interactions of pharmacological agents with pituitary hormone release, on the one hand, and the modulation of hormone release in pathological conditions, on the other hand. Perinatal transient hazards may induce permanent alterations in adaptive behavior when tested in adult age. Corticosteroid-induced deviation of avoidance behavioral reactions may be opposed by simultaneous administration of ACTH-like peptides. These observations revealed that a balance of the glucocorticoids and ACTH-like peptides in perinatal period basically determine the adaptative reaction of animals in adult age. Immune system may be called as a mobile brain since its tremendous information capacity and its responsiveness to alterations of chemical environmental signals. Recent data support the view that there is a bidirectional communication between the neuro-endocrine adaptational axis and the immune system. Stress hormones can alter the immune response and mononuclear cells produce factors that change the neuroendocrine regulation. In addition to these, prohormones are synthesized in mononuclear cells that may be involved in regulation of signalization between cells and in activation of endocrine system and brain functions.     

Alterations in antigenic structure of gonadotropins following deglycosylation

Radioimmunological techniques were utilized to probe possible changes in conformation of gonadotropins (human chorionic gonadotropin-hCG; and ovine luteinizing hormone—oLH) following chemical deglycosylation (DG-hCG and DG-LH). All antisera produced in rabbits, rats or mice contained antibodies that were specific to the deglycosylated hormones with the native hormones showing weak and non-parallel cross-reaction (<5%), but with rabbit antibodies to native hormones the deglycosylated hormones were fully reactive. Using hCG, asialo-hCG (A-hCG) and DG-hCG, we have shown that removal of sugars internal to sialic acid is required to produce these specific antibodies. These are in complete agreement with the observations that extensive deglycosylation of these hormones is necessary to induce changes in biological activity at the cellular level. Based on these data, we suggest that chemical deglycosylation results in changes in antigenic structure of these hormones by generation of new determinants or exposure of previously buried sites and these changes are of no consequence to receptor recognition.     

Nonopiate active proenkephalin-derived peptides are secreted by T helper cells

Recent investigations have shown that the neuroendocrine and immune systems profoundly affect each other. In part, these interactions occur via common chemical messengers and receptors. One possible shared chemical messenger is the opioid precursor preproenkephalin, for which high concentrations of messenger RNA are present in brain, adrenal, and activated T helper cells. Because the biologic action of most peptide messengers depends on the posttranslational processing of the precursor, we have examined T helper cell lines for the production of proenkephalin-derived peptides. These peptides were characterized by multiple radioimmunoassays, gel filtration chromatography, and opiate radioreceptor assays. We found that activated T helper cells secrete significant concentrations of high-molecular-weight, opiate-inactive peptides, which are distinct from the proenkephalin-derived peptides of the neuroendocrine system. These studies clearly indicate cell-specific processing of proenkephalin, and suggest that the T helper cell-secreted products may have nonopiate receptor-mediated actions.     

Immunohistological localization of regulatory peptides in the midgut of the female mosquitoAedes aegypti

The midgut of the female mosquitoAedes aegypti was studied immunohistologically with antisera to various regulatory peptides. Endocrine cells immunoreactive with antisera to perisulfakinin, RFamide, bovine pancreatic polypeptide, urotensin 1, locustatachykinin 2 and allatostatins A1 and B2 were found in the midgut. Perisulfakinin, RFamide and bovine pancreatic polypeptide all react with the same, about 500 endocrine cells, which were evenly distributed throughout the posterior midgut, with the exception of its most frontal and caudal regions. In addition, these antisera recognized three to five neurons in each ingluvial ganglion and their axons, which ran longitudinally over the anterior midgut, as well as axons innervating the pyloric sphincter. The latter axons appear to be derived from neurons located in the abdominal ganglia. Antisera to two different allatostatins recognized about 70 endocrine cells in the most caudal area of the posterior midgut and axons in the anterior midgut whose cell bodies were probably located in either the brain or the frontal ganglion. Antiserum to locustatachykinin 2 recognized endocrine cells present in the anterior midgut and the most frontal part of the posterior midgut, as well as about 50 cells in the most caudal region of the posterior midgut. Urotensin 1 immunoreactivity was found in endocrine cells in the same region as the perisulfakinin-immunoreactive cells, but no urotensin-immunoreactive axons were found in the midgut. Double labeling experiments showed that the urotensin and perisulfakinin immunoreactivities were located in different cells. Such experiments also showed that the locustatachykinin and allatostatin immunoreactivities in the most caudal area of the posterior midgut were present in different cells. No immunoreactivity was found in the mosquito midgut when using antisera to corazonin, allatotropin or leucokinin IV. Since these peptides have either been isolated from, or can reasonably be expected to be present in mosquitoes, it was concluded that these peptides are not present in the mosquito midgut.     

Immunocytochemical localization of cathepsins B, H, and L in the rat gastro-duodenal mucosa

Cathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.     

Identification of Immunoreactive Material in Mammoth Fossils

The fossil record represents a history of life on this planet. Attempts to obtain molecular information from this record by analysis of nucleic acids found within fossils of extreme age have been unsuccessful or called into question. However, previous studies have demonstrated the long-term persistence of peptides within fossils and have used antibodies to extant proteins to demonstrate antigenic material. In this study we address two questions: Do immunogenic/antigenic materials persist in fossils? and; Can fossil material be used to raise antibodies that will cross-react with extant proteins? We have used material extracted from a well-preserved 100000-300000-year-old mammoth skull to produce antisera. The specificity of the antisera was tested by ELISA, western blotting, and immunohistochemistry. It was demonstrated that antisera reacted specifically with the fossils and not the surrounding sediments. Reactivity of antisera with modern proteins and tissues was also demonstrated, as was the ability to detect evolutionary relationships via antibody-antigen interactions. Mass spectrometry demonstrated the presence of amino acids and specific peptides within the fossil. Peptides were purified by anion-exchange chromatography and sequenced by tandem mass spectrometry. The collagen-derived peptides may have been the source of at least some of the immunologic reactivity, but the antisera identified molecules that were not observed by mass spectrometry, indicating that immunologic methods may have greater sensitivity. Although the presence of peptides and amino acids was demonstrated, the exact nature of the antigenic material was not fully clarified. This report demonstrates that antibodies may be used to obtain information from the fossil record.     

Impact of sex hormones, insulin, growth factors and peptides on cartilage health and disease

Sex hormones contribute to the pathogenesis of osteoarthritis (OA) in both sexes. OA is normally not seen in pre-menopausal women, whereas men may develop the disease as early as the 30th year of life. OA also shows increased incidence in association with diseases such as diabetes mellitus. Recent years have seen characterization of essential components of a functional endocrinal network in the articular cartilage comprising not only sex hormones but apparently insulin, growth factors and various peptides as well. In this review, we summarize the latest information regarding the influence of sex hormones, insulin, growth factors and some peptides on healthy cartilage and their involvement in osteoarthritis. Both animal and human research data were considered. The results are presented in an information matrix that identifies what is known, with supporting references, and identifies areas for further investigation.     

Immunocytochemical localization of cathepsins B,H, and L in the rat gastro-duodenal mucosa

Summary Cathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.     

The endocrine cells in the gastroenteropancreatic system of the bowfin, Amia calva L.: an immunohistochemical, ultrastructural, and immunocytochemical analysis.

The gastroenteropancreatic (GEP) endocrine system of bowfin (Amia calva) was described using light and electron microscopy and immunological methods. The islet organ (endocrine pancreas) consists of diffusely scattered, mostly small islets and isolated patches of cells among and within the exocrine acini. The islets are composed of abundant, centrally located B cells immunoreactive to bovine and lamprey insulin antisera and D cells showing a widespread distribution and specificity to somatostatin antibodies. A and F cells are present at the very periphery of the islets and are immunoreactive with antisera against glucagon (and glucagon-like peptide) and several peptides of the pancreatic polypeptide (PP)-family, respectively. The peptides of the two families usually collocates within the same peripheral islet cells and are the most common immunoreactive peptides present in the extra-islet tissue. Immunocytochemistry and fine structural observations characterised the granule morphology for B and D cells and identified two cell types with granules immunoreactive to glucagon antisera. These two putative A cells had similar granules, which were distinct from either B or D cells, but one of the cells had rod-shaped cytoplasmic inclusions within cisternae of what appeared to be rough endoplasmic reticulum. The inclusions were not immunoreactive to either insulin or glucagon antisera. Only small numbers of cells in the stomach and intestine immunoreacted to antisera against somatostatin, glucagon, and PP-family peptides. The paucity of these cells was reflected in the low concentrations of these peptides in intestinal extracts. The GEP system of bowfin is not unlike that of other actinopterygian fishes, but there are some marked differences that may reflect the antiquity of this system and/or may be a consequence of the ontogeny of this system in this species.     

AR4-2J cells: A model to study polypeptide hormone receptors

The AR4-2J cell line is derived from a transplantable tumour of the exocrine rat pancreas. Acinar in origin, this cell line contains significant amounts of amylase and can be grown in continuous culture. Manyin vitro studies have been done using these cells; these studies were often complemented within vivo experiments on animals. Particularly, many polypeptide hormones interacting with specific receptors located on the cell membrane have been analysed. The accurate knowledge of the hormone-receptor interactions has allowed to design interesting analogs of these hormones. In several cases, these compounds are powerful antagonists and are able to control cell proliferation induced by the corresponding polypeptide hormones. Other cell lines are useful to understand human pancreatic cancer. These human cell lines (Capan-1, Panc-1 for example) are of ductal origin and differ from AR4-2J cells, especially regarding the distribution of several polypeptide hormone and growth factor receptors. Both models are important for basic studies of neuropeptides, gastrointestinal peptides and their receptors, as well as for a better understanding of the underlying mechanisms of human pancreatic cancer.     

Mast cell heterogeneity: effects of neuroenteric peptides on histamine release

Recent reports suggesting that the actions of certain neuroenteric peptides may be mediated in part by the secretion of histamine and other mast cell contents could have important implications for gastrointestinal motility and secretion. However, evidence for a mast cell-hormonal interaction is based on studies using peritoneal or cutaneous mast cells. Because intestinal mucosal mast cells (MMC) differ functionally from peritoneal mast cells (PMC), we compared the effects of several neurotransmitters and intestinal hormones on histamine secretion from two mast cell types in the rat. MMC hyperplasia was induced in rats by infection with the nematode Nippostrongylus brasiliensis, and MMC were isolated from the small intestine by collagenase digestion. Substance P, somatostatin, vasoactive intestinal polypeptide (VIP), neurotensin, and bradykinin had a potent secretagogue effect on (10(-7) to 10(-4)M) PMC which was temperature-, energy-, and calcium-dependent. In contrast to PMC, MMC released significant amounts of histamine only when challenged with substance P. Acetylcholine, bombesin, motilin, and pentagastrin had no secretory effect on either PMC or MMC. The differences between PMC and MMC in responsiveness to peptides could not be attributed to the MMC isolation procedure because PMC treated similarly or mixed with MMC suspensions retained their responsiveness to these stimuli. Our results extend the concept of neurocrine control of mast cell function, but indicate that mast cells from different sites have distinct profiles of responsiveness to regulatory peptides.     

Immunocytochemistry of secretory peptides: immunochemical dissections of peptide diversity

Immunocytochemistry of secretory peptides involves the localization of hormonal, neuronal, and paracrine messenger molecules at the cellular level. Immunological studies of these peptides have revealed them to be heterogenous at both the cellular, subcellular, ontogenetic, phylogenetic, and molecular level. It is probable that this heterogeneity reflects a diversity in function and that the same peptide backbone may be utilized for different tasks by different cells. One tentative way in which this could be brought about is by selective proteolytic, processing mechanisms. Although preliminary evidence indicates that such site-dependent processing occurs, much work remains to be done on the accurate localization of peptide fragments by specific, precise, sensitive, and selective tools. Recent progress in antibody production and evaluation as well as in immunocytochemical methodology has put such tools at our disposal. The present review discusses the use of these tools with particular emphasis on the interpretation of results, and their correlation to chemical and physiological information.     

Immunochemical studies of (Na+ + K+)-ATPase using site-specific, synthetic peptide directed antibodies

Rabbit antisera were raised against a series of synthetic peptides corresponding to regions of the alpha subunit of lamb kidney (Na+ + K+)-ATPase which chemical labeling studies and hydropathy plots of the amino-acid sequence suggest are exposed, accessible regions of the enzyme and may comprise the cation selectivity region, the ATP and cardiac glycoside binding sites, and the phosphorylation site. Five of six peptides tested (11-15 residues in length) were immunogenic and the antisera to four peptides recognized the intact, electroblotted (Western blot analysis) alpha subunit. Immunization with peptides conjugated to keyhole limpet haemocyanin (KLH) produced antipeptide antibodies for seven of nine conjugates. Antisera to four peptide conjugates recognized the native enzyme, confirming predictions that these sequence regions are exposed regions of the holoenzyme. In addition, a collection of four polyclonal antisera and five monoclonal antibodies raised to native holoenzyme were tested for their ability to bind to the peptide conjugates. In this way, two NH2-terminal sequence regions (1-12 and 16-30) and the putative ATP-binding site region (496-506) were identified as epitopes of the native enzyme. These results confirm some aspects of the transmembrane folding models proposed by Shull et al. and Kawakami et al. for the membrane-bound (Na+ + K+)-ATPase.     

Immunohistochemical investigations of neuropeptides in the brain,corpora cardiaca,and corpora allata of an adult lepidopteran insect,Manduca sexta (L)

In the brain of adult specimens of the tobacco hornworm moth, Manduca sexta (L), cells immunoreactive for several kinds of neuropeptides were localized by means of the PAP procedure, by use of antisera raised against mammalian hormones or hormonal peptides. In contrast, no such neurosecretory cells were found in the corpora cardiaca and corpora allata (CC/CA); in the CC/CA, however, immunoreactive nerve fibres were observed, reaching these organs from the brain. The neurosecretory cells found in the brain were immunoreactive with at least one of the following mammalian antisera, namely those raised against the insulin B-chain, somatostatin, glucagon C-terminal, glucagon N-terminal, pancreatic polypeptide (PP), secretin, vasoactive intestinal polypeptide (VIP), glucose-dependent insulinotropic peptide (GIP), gastrin C-terminus, enkephalin, alpha- and beta-endorphin, Substance P, and calcitonin. No cells were immunoreactive with antisera specific for detecting neurons containing the insulin A-chain, nerve growth factor, epidermal growth factor, insulin connecting peptide (C-peptide), polypeptide YY (PYY), gastrin mid-portion (sequence 6-13), cholecystokinin (CCK) mid-portion (sequences 9-20 and 9-25), neurotensin C-terminus, bombesin, motilin, ACTH, or serotonin. All the neuropeptide-immunoreactive cells observed emitted nerve fibers passing through the brain to the CC and in some cases also to the CA. In CC these immunoreactive nerve fibers tended to accumulate near the aorta. It was speculated that neuropeptides are released into the circulating haemolymph and act as neurohormones.     

Epitope peptides of influenza H3N2 virus neuraminidase gene designed by immunoinformatics

The virus surface protein neuraminidase (NA) is a main subtype-specific antigen in influenza type A viruses. Neuraminidase functions as an enzyme to break the bonds between hemagglutinin (HA) and sialic acid to release newly formed viruses from infected cells. In this study, NA genes from the H3N2 subtype virus were sequenced and NA proteins were screened for B-cell epitopes and assessed based on immunoinformatics. Based on this information, three peptides ES8, RR9, and WK7 (covering amino acid residues 221-228, 292-300, and 383-389, respectively) of the NA protein were selected and synthesized artificially. These peptides were used to immunize New Zealand rabbits subcutaneously to raise antisera. Results showed that these three peptides were capable of eliciting antibodies against H3N2 viruses in a specific and sensitive manner, detected in vitro by enzyme-linked immunosorbent assay. Furthermore, hemadsorption anti-releasing effects occurred in three antisera mixtures at a dilution of 1:40. Alignment using database software showed that amino acid residues in these three epitope peptides were substituted at specific sites in all the NAs sequenced in this study. We suggest that these NA epitope peptides might be used in conjunction with HA proteins as vaccine antigens.     

Immunocytochemical localization of peptidergic neurons and neurosecretory cells in the neuro-endocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides

A large number of antisera to regulatory vertebrate peptides was tested immunocytochemically on the nervous system of the Colorado potato beetle to further characterize the peptidergic cells of the neuro-endocrine system and to reveal cells participating in endocrine control mechanisms. Neurons, neurosecretory cells, axons and axon terminals were revealed by antisera to ACTH, gastrin, CCK, alpha-endorphin, beta-endorphin, gamma 1-MSH, insulin, motilin, human calcitonin, growth hormone, somatostatin, CRF, ovine prolactin and rat prolactin. Together with previously described results these findings demonstrate that at least 19 different peptidergic cell types are present in the Colorado potato beetle. Several of these cell types are identical with the known neurosecretory cells, while others have not been identified before. The functions of the immunoreactive neurons are as yet unclear, although in two cases the localization of these cells gives some clues. Thus the lateral neurosecretory cells, which are immunoreactive with antisera to beta-endorphin and ovine prolactin, may regulate corpus allatum activity, whereas a CRF immunoreactive substance seems to be used as neurotransmitter by antennal receptors. These immunocytochemical findings do not imply that the immunoreactive substances are evolutionarily related to the vertebrate peptides to which the antisera were raised. It is postulated that if the part of the substance recognized by a certain antiserum is functionally important for the insect, which should be so if the insect peptide is evolutionarily related to its vertebrate homologue, the antiserum should reveal homologous cells in different insect species. The consequence of this hypothesis is, that if an antiserum does not reveal homologous neurons in different insect species, the immunologically demonstrated substance is probably of little physiological importance, and will not be related evolutionarily to the vertebrate analogue. The positive immunocytochemical results in the Colorado potato beetle are discussed in relation to these considerations.     

Differential location of peptide hormones in the secretory pathway of insect adipokinetic cells

Immunoreactivity of granules containing secretory material in the adipokinetic cells of the insect Locusta migratoria was studied using antisera specific for the adipokinetic hormone-associated peptides (AAP) I, II and III. Immunocytochemical detection of these associated peptides represents a new strategy for studying the intracellular location of the adipokinetic hormones and their prohormones. Fixation with 2% glutaraldehyde and 2% formaldehyde with low-temperature embedding in Lowicryl HM20 allowed highly selective immunogold labelling of both secretory and intracisternal granules. All three associated peptides were co-localized in secretory granules. This indicates that also all three adipokinetic hormones can be co-localized in these granules, which was confirmed by experiments in which, after secretory stimulation, adipokinetic hormone III was released from the adipokinetic cells together with adipokinetic hormones I and II. The immunopositivity of the intracisternal granules was similar to that of the secretory granules, although with the exception that the intracisternal granules did not show any specific reaction with anti-AAP III. The presence of AAP I and AAP II in intracisternal granules indicates that these granules only function as stores of adipokinetic prohormones I and II and not of adipokinetic prohormone III. The observed differences in storage in intracisternal granules among the three adipokinetic prohormones suggest differences in physiological significance of the three adipokinetic hormones in L. migratoria.