Localization Of Galanin Research Articles

Colorectal cancer patients exhibit increased levels of galanin in serum and colon tissues.

Galanin (GAL) is a 30-amino acid neuropeptide that is expressed in both the central and peripheral nervous system, including the enteric nervous system (ENS). Increased GAL concentrations have been identified in the blood of colorectal cancer (CRC) patients. The aim of the present study was to assess whether sections of the colon wall containing ENS plexuses or CRC tumor are associated with increased GAL concentrations. Blood samples were collected from 68 CRC patients and 39 healthy volunteers. In addition, samples of CRC tumors and colon wall tissue in close proximity to and distant from the neoplastic tissue were obtained from 22 CRC patients. The GAL concentration of sera and tissue homogenates obtained from three sections of the colon wall (mucosa with submucosa, muscularis externa and CRC tumor) was analyzed by ELISA. The localization of GAL was evaluated using immunohistochemistry and morphometry was used to measure the distribution of GAL-immunoreactive (GAL-Ir) myenteric plexuses in the vicinity of cancer invasion and in sections of the colon wall distant from the tumor. The GAL serum concentration of CRC patients was 2.4 times higher than that of the control group. The GAL concentration was highest in the homogenates of neoplastic tissue and mucosa obtained from the control (distant) section of the intestinal wall, followed by that in the mucosa and muscularis externa proximal to the tumor. The lowest GAL concentrations were identified within the muscular layer of the colon wall located distant from the tumor. Strong GAL immunoreactivity was identified in the cancer cells, intestinal epithelium and the submucosal and myenteric plexuses. Morphometric analysis revealed that the GAL-Ir myenteric plexuses in the vicinity of tumor infiltration were significantly smaller in size than those in the intact section of the large intestine. Furthermore, no associations were identified between the clinicopathological characteristics of CRC patients and GAL serum and tissue concentration. The increased GAL serum concentrations observed in CRC patients in comparison to healthy controls may result from GAL secretion by CRC tumors, however, other sources of GAL cannot be excluded. The atrophy of myenteric plexuses within close proximity to the tumor may affect the colon function of CRC patients. In conclusion, investigation into the presence of GAL in the colon wall and serum of CRC patients revealed that serum and tissue GAL levels may present useful potential biomarkers in CRC patients.

Expression of galanin and galanin receptor-1 in normal bone and during fracture repair in the rat

The neuropeptide galanin (GAL) has recognized physiological actions in the nervous system and other tissues, but there is no documented evidence of GAL influencing normal or pathological bone metabolism. GAL expression, however, is upregulated in central and peripheral nerves following axotomy and is known to influence neural regeneration. Thus, severance of skeletal-associated nerves during fracture could similarly increase local GAL concentrations and thereby influence fracture healing. The initial aim of this study was therefore to identify the presence of GAL in normal bone and/or fracture callus by assessing the concentration and cellular localization of GAL in intact and/or fractured rat rib, using radioimmunoassay and immunohistochemistry, respectively. Groups of Sprague–Dawley rats (13 weeks old) had their left sixth ribs surgically fractured or underwent sham surgery and then calluses and nonfractured rib samples were analyzed at 1 and 2 weeks postsurgery ( n = 5–6 per group). Low (basal) concentrations of GAL were detected in control ribs, whereas at 1 and 2 weeks postfracture, callus samples contained markedly increased levels of peptide (∼32- and 18-fold increase, respectively, relative to controls; P < 0.01), revealing a strong upregulation during bone healing. Plasma GAL concentrations were also increased at 2 weeks postfracture ( P < 0.005). In normal (nonfractured) rib, minimal levels of GAL-like immunoreactivity (LI) were present in cortical bone, periosteum, endosteum, and surrounding skeletal muscle. In costal cartilage plates, intense GAL-LI was present in all chondrocytes of the hypertrophic zone and in a population of chondrocytes in the reserve zone. GAL-LI was not present, however, in chondrocytes in the proliferative zone of costal cartilage or skeletal muscle fibers. In fracture callus, levels of GAL-LI were moderate to intense in osteoprogenitor cells and osteoblasts, in some chondrocytes, and in cartilaginous, osseous, and periosteal matrices. Subsequent studies revealed the presence of galanin receptor-1-like immunoreactivity (GALR1-LI) in most cell types shown to contain GAL-LI, although the distribution of GALR1-LI was more extensive in reserve zone chondrocytes than that of GAL-LI; and GALR1-LI also appeared in late proliferative zone chondrocytes of costal cartilage. In summary, GAL concentrations were significantly increased in fracture callus and plasma of rats that underwent rib fracture. In addition, GAL- and GALR1-LI was also detected in specific cells and structures within costal cartilage, bone, and fracture callus. These results strongly implicate GAL in aspects of cartilage growth plate physiology and fracture repair, possibly acting in an autocrine/paracrine fashion via GALR1.

Localization of the neuropeptide galanin in nerve fibers and epithelial keratinocytes of the rat molar gingiva.

Knowledge of the histochemical substrates of cellular and neurovascular connections in the gingiva is essential in order to understand the initial mechanisms of inflammation in the periodontium. Since the localization of the neuroendocrine peptide galanin in the gingiva is still unclear, we used immunohistochemical, in situ hybridization and immunoblot techniques to assess the localization of galanin in the gingiva of rat molars. Galanin-immunoreactive nerve fibers were located around blood vessels in the lamina propria, beneath the epithelium, in the epithelial-proprial junction and in the basal layer of the epithelium. Galanin was highly expressed in the suprabasal keratinocytes of the gingival epithelium. The localization of galanin in gingival nerve fibers and the expression of galanin in keratinocytes of the gingival epithelium indicate that galanin may be a possible regulator of different cellular functions in the gingiva.

Monoclonal antibody to rat galanin: production, characterization, and in vivo immunoneutralization activity.

A monoclonal antibody (MAb) to galanin was prepared by cell fusion of myeloma Fox-NY and spleen cells from Robertsonian mice immunized with rat galanin. Hybridomas producing high-affinity antibodies were cloned in pristine-primed Balb/c mice. The antibody was purified by affinity chromatography and concentrated to 12 mg IgG/mL by dialysis. Immunoreactivity of the antibody was screened by radioimmunoassay. Ascites fluid contained approximately 10 mg/mL IgG that belong to the subclass of IgG2a as determined by enzyme-linked immunoadsorbent assay (ELISA). The titer of this IgG2a antibody entitled #G65G was 1:10,000 and the ID50 for rat galanin was 1000 fmol/mL as determined by liquid phase radioimmunoassay. Immunohistochemistry showed that this galanin MAb stains densed, beaded processes distributed to the enteric plexuses, where they appear to encircle neuronal cell bodies, to the muscle layer, where they are particularly abundant in the circular muscle layer and in the deep muscular layer, and to the mucosa. In vivo capacity of immunoneutralization by this antibody was tested in male Sprague-Dawley rats fasted for 24 h and anesthetized with urethane. Systemic injection of protein A purified galanin antibody (6 mg/rat) decreased by 70% of the inhibitory effect of intravenous galanin (2 nmol/kg/h i.v.) on gastric acid secretion induced by intracisternal TRH analog. These results show that galanin antibody #G65G is useful for in vivo immunoneutralization of galanin effects and is a valuable tool for immunohistochemical localization of galanin in gastrointestinal tissues.

Localization of galanin and its binding sites in the quail brain

Avian galanin was first isolated from the chicken intestine, and subsequently we isolated the same peptide from the quail oviduct. Galanin is known to be a multifunctional peptide in the neuroendocrine system in mammals, while little information is available for the galanin action in the central nervous system in birds. In the present study, therefore, we examined the presence and the localization of galanin and its binding sites in the quail brain. The binding of [ 125I]iodo-avian galanin to brain tissues specifically inhibited as a function of the concentrations of unlabeled avian galanin. In all of the observed brain regions, the number of galanin-binding sites when compared on the basis of unit weight was maximal in the interbrain including the preoptic and hypothalamic regions and minimal in the cerebellum. The Scatchard plot analysis of the galanin binding to the interbrain revealed that the apparent equilibrium constant of dissociation ( K d was 0.281 (95% confidence interval, 0.234-0.351) nM, suggesting the presence of a single class of high-affinity binding sites. Although galanin-like immunoreactivity was found in several restricted regions throughout the brain, the most intense immunoreaction was present in the diencephalic region. Abundant immunoreactive cell bodies and/or fibers were localized in several preoptic and hypothalamic nuclei, i.e., the nucleus preopticus medialis (PONI), the nucleus paraventricularis (PVN), the nucleus periventricularis hypothalami (PHN) and the nucleus infundibuli (IN), that are known to be involved in the control of reproductive behaviors as well as the pituitary hormone secretion. A substantial number of immunoreactive cell bodies and/or fibers was further observed within the septal nuclei in the telencephalon and several mesencephalic nuclei including the nucleus intercollicularis (ICo) involved in the control of vocalizations. These results suggest that avian galanin may act as an important factor to modulate the some reproductive behaviors and/or the pituitary hormone secretion in the bird.

Localization of galanin and its binding sites in the quail brain

Avian galanin was first isolated from the chicken intestine, and subsequently we isolated the same peptide from the quail oviduct. Galanin is known to be a multifunctional peptide in the neuroendocrine system in mammals, while little information is available for the galanin action in the central nervous system in birds. In the present study, therefore, we examined the presence and the localization of galanin and its binding sites in the quail brain. The binding of [ 125 I]iodo-avian galanin to brain tissues specifically inhibited as a function of the concentrations of unlabeled avian galanin. In all of the observed brain regions, the number of galanin-binding sites when compared on the basis of unit weight was maximal in the interbrain including the preoptic and hypothalamic regions and minimal in the cerebellum. The Scatchard plot analysis of the galanin binding to the interbrain revealed that the apparent equilibrium constant of dissociation ( K d was 0.281 (95% confidence interval, 0.234-0.351) nM, suggesting the presence of a single class of high-affinity binding sites. Although galanin-like immunoreactivity was found in several restricted regions throughout the brain, the most intense immunoreaction was present in the diencephalic region. Abundant immunoreactive cell bodies and/or fibers were localized in several preoptic and hypothalamic nuclei, i.e., the nucleus preopticus medialis (PONI), the nucleus paraventricularis (PVN), the nucleus periventricularis hypothalami (PHN) and the nucleus infundibuli (IN), that are known to be involved in the control of reproductive behaviors as well as the pituitary hormone secretion. A substantial number of immunoreactive cell bodies and/or fibers was further observed within the septal nuclei in the telencephalon and several mesencephalic nuclei including the nucleus intercollicularis (ICo) involved in the control of vocalizations. These results suggest that avian galanin may act as an important factor to modulate the some reproductive behaviors and/or the pituitary hormone secretion in the bird.

The prepubertal ontogeny of galanin-like immunoreactivity in the male Meishan pig brain

Galanin (GAL) is a neuropeptide found in the mammalian brain and is involved in numerous functions including the control of feeding, growth and reproduction, and therefore may be an important peptide to study in agricultural species. We describe the immunohistochemical localization of GAL throughout prepubertal development in the Meishan pig, a Chinese breed known for its superior reproductive characteristics, but slow growth rate and abundant adipose tissue. Brains of animals from gestational day (g) 30, 50, 70, 90 and 110 and postnatal day (pn) 1, 10, 20 and 50 (duration of pregnancy averaged 114 days) were processed using a standard immunohistochemical technique utilizing a commercially available rabbit anti-porcine GAL antibody. Galanin-like immunoreactivity (GAL-IR) in cell bodies and fibers was evident in the brain at g30, primarily in the hypothalamus. Throughout prenatal development, cell bodies containing GAL-IR generally increased in number and distribution in the brain. During postnatal development, the number of cell bodies displaying GAL-IR decreased, particularly in hypothalamic areas. The distribution of GAL-IR in fibers became more widespread throughout gestational development, showing a pattern by pn1 that continued during later postnatal ages. The intensity of GAL-IR in fibers also increased throughout gestation. Some additional increases in immunoreactivity occurred postnatally, especially in the periventricular hypothalamus. The results of this study indicate that the distribution of GAL-IR in cell bodies and fibers in the Meishan pig brain was similar to that seen in other species, including the rat. These results support the hypothesis that GAL participates in the control of feeding, growth and reproduction in the pig.

Modulation of galanin and neuromedin U-like immunoreactivity in rat corticotropes after alteration of endocrine status

The localization of galanin in rat lactotropes and human corticotropes is well established. Neuromedin U immunoreactivity is present in rat corticotropes but radioimmunoassay of thyroid-manipulated rat pituitaries has also linked it to the thyroid axis. We found galanin immunoreactivity in some rat corticotropes, so we have re-examined rat anterior pituitary galanin- and neuromedin U-like immunoreactivity by use of immunocytochemistry and electron microscopy in rats in the normal state and after estrogen administration or adrenalectomy. In normal rats galanin immunoreactivity was present in a few corticotropes and lactotropes, females showing more than males; neuromedin U-like immunoreactivity was present in some thyrotropes and most corticotropes, in both sexes. Where galanin, neuromedin U and ACTH immunoreactivities were colocalized in corticotropes they were present in the same granules. Estrogen administration caused an increase in number of galanin immunoreactive lactotropes, as previously shown. The proportion of neuromedin U-positive corticotropes was not affected. After adrenalectomy, only females showed a significant increase in the proportion of galanin-positive corticotropes. Neuromedin U immunoreactivity was significantly increased in both sexes, as previously shown. Thus, in rat, as in man, galanin can be present in corticotropes and its expression appears to be sex-related. This finding, and the demonstration of thyrotrope neuromedin U (only examined in normal females), provide correlation with previous experiments. The influence of endocrine status on the expression of these novel peptides underlines the inherent plasticity of pituitary endocrine cells.

Identification of galanin-immunoreactive cells in the anterior pituitary of male monkeys (Macaca fascicularis).

The localization of galanin (GAL) was studied in the anterior pituitary of adult male macaque monkeys immunohistochemically. GAL-immunoreactive fiber bundles were noted in the posterior lobe of the pituitary. Cells immunoreactive for GAL were observed in the anterior lobe, and colocalization studies revealed that GAL-like immunoreactivity was present in gonadotrophs and thyrotrophs. The differences among the monkey, human and rat in GAL-immunoreactive localization may indicate that the regulation of GAL in the three species differs.

Evidence for upregulation of galanin synthesis in rat glial cells in vivo after colchicine treatment

The localization of galanin (GAL) and GAL mRNA was studied in rat brain after colchicine or vinblastine treatment using in situ hybridization and immunohistochemistry. GAL-like immunoreactivity was found in glial cells, presumably activated microglia, in the cortex, caudate nucleus and septum, mainly on the injection side. GAL mRNA expression was found in small cells in the same areas with an overlapping distribution, including the adjacent white matter. The results suggest that the glial cells initiate synthesis of the peptide GAL in response to intraventricular injection of high doses of the 2 mitosis inhibitors.

Colocalization of Galanin and Prolactin Within Secretory Granules of Anterior Pituitary Cells in Estrogen-Treated Fischer 344 Rats*

Galanin is localized within specific cell types of the rat anterior pituitary gland (AP). Immunocytochemical studies at the light microscope level have shown that lactotrophs, somatotrophs, and thyrotrophs contain galanin in the intact female rat, whereas lactotrophs in the male AP do not. We recently reported that galanin and PRL release from estrogen-treated male and female pituitary cells in culture are coregulated by dopamine, TRH, and somatostatin. This suggested that galanin is stored within secretory granules, conceivably with PRL. Using postembedding immunocytochemistry at the ultrastructural level, the objectives of this study were to: 1) determine the subcellular location of galanin in the AP; 2) elucidate if galanin and PRL are colocalized within the same secretory granules; and 3) compare the cellular localization of galanin in the male and female AP. Male and ovariectomized female (OVEX) Fischer 344 rats were implanted with estradiol-containing or empty Silastic capsules for 2 weeks. Postembedding immunogold labeling was performed using rabbit (for galanin) and guinea pig (for PRL) generated antisera. Two different sizes of colloidal gold spheres were used to localize the hormones in the same tissue section. Galanin was primarily localized in secretory granules of adenohypophyseal cells. Based upon immunocytochemical results and morphological criteria, galanin was contained in somatotrophs but not lactotrophs in the male and OVEX AP. The AP of estrogen-treated rats contained more specific immunogold labeling for galanin than untreated rats. The increased immunoreactivity for galanin was notably associated with lactotrophs. After exposure to estrogen, galanin and PRL were colocalized within the same secretory granules of the male and OVEX pituitary cells. We conclude: 1) galanin is localized within secretory granules of the rat AP; 2) galanin and PRL are colocalized within secretory granules of the male and OVEX AP after estrogen treatment; and 3) galanin is localized in similar cell types in the male and OVEX AP, before and after estrogen treatment. These data provide a morphological basis for the coregulation of galanin and PRL secretion by hypothalamic factors.

Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence

The role of the neuropeptide galanin (GAL) in rat hypothalamus has been studied in different experimental models. Thus, the effect of GAL on potassium-induced dopamine release was analyzed in vitro, and the localization of GAL and GAL binding sites was studied with immunohistochemistry and receptor autoradiography, respectively. In the median eminence GAL and presumably dopamine were found to coexist in nerve endings and this area contained a high density of 125I-GAL binding sites. In vitro experiments revealed that GAL inhibited the release of [ 3H]dopamine in a dose-dependent manner (IC 50=7 10 nM), possibly via a presynaptic receptor.

Distribution of galaninlike immunoreactivity in the rat central nervous system

The localization of galanin (GAL) immunoreactive (IR) neuronal structures in the rat central nervous system has been investigated by using the indirect immunofluorescence technique. GAL-IR structures were seen in high concentrations in the hypothalamus, medulla oblongata, and spinal cord. Less extensive systems were detected in the telencephalon, thalamus, mesencephalon, and pons, while virtually no GAL-positive structures were seen in the olfactory bulb and cerebellum. Major populations of cell bodies staining for GAL-like material were seen in many areas. In the telencephalon somata were revealed in the bed nucleus of stria terminalis, in the nucleus of the diagonal band, medial septum, and in the medial aspects of the central amygdaloid nucleus, and in small numbers in cortical areas. The anterodorsal and periventricular nuclei of the thalamus contained positive cell bodies. In the hypothalamus GAL-IR somata were seen in the medial and lateral preoptic nuclei, arcuate nucleus, periventricular nucleus, in the dorsomedial nucleus, in the medial forebrain bundle area, in the tubular, caudal, accessory, supraoptic, and paraventricular magnocellular nuclei and lateral to the mammillary recess. The dorsal raphe nucleus hosted a large number of GAL-positive somata. Locus coeruleus of the pons contained a large number of GAL-IR perikarya. In the medulla oblongata positive somata were found in the caudal spinal trigeminal nucleus, the nucleus of the solitary tract, and in the ventral lateral area just rostral to area postrema. Small cell bodies were detected in the superficial layers of the dorsal horn of the spinal cord at all levels and in lamina X at lumbar levels. Analysis of GAL-positive fibers in the telencephalon revealed highly or medium-dense networks in the lateral septal nucleus, in the bed nucleus of stria terminalis, and in the central and medial amygdaloid nuclei. Positive fibers were found in the thalamus in and around the periventricular nucleus as well as in the lateral habenular nucleus and extending in a lateral, caudal direction from the third ventricle and fasciculus retroflexus to the lateral tip of the medial lemniscus. In the hypothalamus the external layer of the median eminence contained a very dense fiber network. Dense or medium-dense GAL-IR networks were detected in the periventricular nucleus, throughout the medial and lateral preoptic areas, in the medial forebrain bundle area, in the dorsomedial nucleus, and lateral to the mammillary recess. In the pons GAL-IR fibers were seen in the parabrachial nuclei, dorsal to the superior olive, and in the periaqueductal central gray.(ABSTRACT TRUNCATED AT 400 WORDS)