Calcitonin gene-related peptide and its mRNA in pulmonary neuroendocrine cells and ganglia

Summary The occurrence of calcitonin gene-related peptide (CGRP) and it's mRNA was studied in lungs of rats and piglets using in situ hybridization with two synthetic oligonucleotide probes followed by immunocytochemistry (ICC). CGRP mRNA was present in pulmonary neuroendocrine cells (PNEC) of both the solitary type and cluster type (neuroepithelial body; NEB) at all levels of the airway epithelium from bronchi to alveoli. The distribution of labelled cells was similar to that previously described with ICC. The 44-mer probe provided stronger hybridization signal than the 34-mer and the two combined increased labelling slightly. Formalin fixation reduced labelling and tended to increase background. Labelling for CGRP mRNA was evenly distributed over the cytoplasm, whereas CGRP-like immunoreactivity (LI) usually was of highest intensity toward the base of the PNEC, suggesting basal accumulation of synthesized peptide. CGRP-LI was also observed in occasional rat ganglia and in some, but not all, piglet ganglia. These local neurons may contribute to the CGRP fibers of airways and vasculature, and could theoretically bridge their dendrites and axons between NEB and the effector organ (e.g. artery or arteriole) thus accomplishing a function similar to the postulated axon reflex.     

Hybridocytochemical detection of mRNA for calcitonin,CGRP, NPY and somatostatin in thyroid parafollicular (C) cells in three rodent species

The present study was aimed at hybridocytochemical (HCC) detection and interspecies comparison of mRNA for calcitonin (CT), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and somatostatin (SS) in thyroid C cells of two rodent families of wild Microtidae: pine voles and common voles and also of laboratory Muridae, Wistar rats. Studies were performed on adult males. The HCC method in situ and immunomax technique were used to detect mRNA. DNA oligonucleotide probes labeled with digoxigenin were used in the HCC method. The obtained results were compared to the results of immunocytochemical (ICC) examinations, where rabbit or mouse antibodies against human CT, SS, NPY and rat CGRP, as well as chromogranin A were performed. In the present study, HCC reaction has demonstrated the presence of mRNA for CT and CGRP in all thyroid C cells in all the species examined. However, mRNA for NPY and SS was observed in very few C cells in rat and in many more C cells in the two species of wild rodents. The distribution of the positive cells corresponded with that of ICC detected cells.     

Calcitonin gene-related peptide in hamster lung and its coexistence with serotonin: a chemical and immunocytochemical study

The mammalian lung may have an important endocrine function besides being involved in gas exchange mechanisms. A number of peptide hormones have been localized to neurons and endocrine cells in the lung where they may contribute to the regulation of local pulmonary functions. We have investigated the presence of calcitonin gene-related peptide (CGRP), in the hamster lung by radioimmunoassay and by immunocytochemistry. Measurable quantities of CGRP were detected in lung tissue. Females had higher lung tissue levels of CGRP-like immunoreactivity (IR) than males. This was not reflected in an observable increase in the intensity or distribution of CGRP-like reactivity with immunocytochemistry. Distinct CGRP-like IR was recorded in clustered (NEB) and solitary (NEC) neuroendocrine cells in neonates, weanlings and adults, including all airways from trachea (NEC only) to bronchi, bronchioles, and alveolar ducts to the level of alveoli (NEC and NEB). In adult hamsters, there seemed to be fewer immunoreactive cells, although intensity was unchanged. In addition some NEB contained serotonin-like IR, and colocalization of the peptide and the amine was noted within some cells. Intra-epithelial beaded nerve fibers, subepithelial fibers, and large-caliber nerves in the hilus region and tracheal wall were also CGRP-IR, and immunoreactive nerves were occasionally found in close association with NEB at the basal pole. Positive nerve fibers were not observed in vessels within the lung, and were sparse in the adventitia of tracheal arteries.     

Neuronal developmental marker FORSE-1 identifies a putative progenitor of the pulmonary neuroendocrine cell lineage during lung development.

The FORSE-1 (forebrain-surface-embryonic) monoclonal antibody (MAb) recognizes a carbohydrate cell surface epitope related to the Lewis-X (LeX) and stage-specific embryonic antigens (SSEAs). In the developing CNS, the FORSE-1 epitope is believed to serve as a marker of progenitor cells. We studied the expression of the FORSE-1 epitope in pulmonary neuroendocrine cells (PNECs) and related neuroepithelial bodies (NEBs), cell types implicated in paracrine regulation of lung development. We used dual immunolabeling to identify PNECs/NEBs in tissue sections from developing rabbit fetal lungs and corresponding primary lung cell cultures. During the early stage (E16), the FORSE-1 MAb labeled primitive airway epithelium, whereas serotonin (5HT) immunoreactivity, a marker of PNEC/NEB differentiation, was negative. After E18, FORSE-1 labeling became restricted to PNECs and NEBs, identified by co-expression with 5HT, then decreased coincident with an increase in 5HT. Expression of the FORSE-1 epitope correlated inversely with 5HT expression in PNEC/NEB cells. FORSE-1 immunoreactivity correlated with cell proliferation assessed by BrdU labeling. Downregulation of the FORSE-1 epitope correlated with maturation of PNECs/NEBs. The presence of few FORSE-1/5HT-positive cells in postnatal lung suggests retention of progenitors. The FORSE-1 epitope was associated with a high molecular weight (286 kD) glycoprotein that decreased with increasing gestational age, as demonstrated by immunoblotting. Overall expression of SSEA-1, -3, and -4 antigens was similar to FORSE-1/5HT, although the former was preferentially localized to neurite-like processes. Because the role of the FORSE-1 epitope in the CNS probably involves cell adhesion and differentiation, we propose a similar function in developing lung. The demonstration of LeX/SSEA antigen expression in the PNEC/NEB cell lineage underscores the importance of these cells in developing lung. Furthermore, the FORSE-1 antigen may identify committed progenitors of the PNEC/NEB cell system.     

Combined non-radioactive detection of peptide hormones and their mRNAs in stomach somatostatin cells

Non-radioactive in situ hybridization (ISH) and immunocytochemistry (ICC) have been used to detect somatostatin (SS) messenger RNA (mRNA) and peptide in antropyloric mucosa of the stomach in the rats. We have applied a method of non-radioactive in situ hybridization histochemistry using digoxigenin labelled oligonucleotide probes to detect somatostatin gene expression in the stomach. In prehybridization stage we used proteinase K (PK) in various concentrations (from 1 to 10 micrograms/ml) and periods (from 10 min to 1 h) but we maintained high background. However it was possible to detect the somatostatin mRNAs in the stomach mucosa making use of either background preventing solutions during the prehybridization, or of levamisole (20 microliters/mg) added into the hybridization buffer or of pepsin. Somatostatin mRNA and peptide signals were scattered all through the mucosa especially localized particularly at the base of the pyloric glands. SS peptide shown by ICC and SS mRNA shown by ISH were observed in different cells.     

血管活性肠肽、降钙素基因相关肽及其受体在气道高反应动物肺内的时空分布

为探讨内源性神经肽在气道高反应性形成中的作用,我们以臭氧应激损伤动物气道上皮细胞,建立气道高反应性动物模型,并观察臭氧应激不同时间肺内血管活性肠肽(vasoactive intestinal peptide,VIP)、降钙素基因相关肽(calcitonin gene-related CGRP)含量变化以及VIP受体(VIPR1)、CGRP受体(GRPR1)mRNA在肺内表达、分布的改变。实验观察到,臭氧应激组动物吸入乙酰甲胆碱后气道阻力高于正常对照组,肺内呈现明显的炎症改变。随臭氧应激时间延长,肺组织匀浆中VIP、CGRP浓度呈先增高后降低的双向改变,CGRP达峰值时间早于VIP。VIPR1、CGRPR1 mRNA表达亦经历了双向过程,VIPR1峰值持续时间长于CGRPR1。在无应激对照组动物,肺间质、支气管上皮细胞、血管内皮细胞、平滑肌细胞均有VIPR1、CGRPR1 mRNA表达。随应激时间的延长,阳性细胞呈斑片状集中于气管、血管周围,染色强度增加,至臭氧应激第8天,阳性染色细胞减少。因此我们推测,臭氧应激可以诱导动物气道高反应性的形成。在炎症的早期,以CGRP的作用为主,与肺损伤早期炎症信号的传递有关,以清除刺激原、及时终止致病原的作用;炎症后期以保护机体、促进修复、减轻损伤为主,VIP发挥主要作用。     

Calcitonin gene-related peptide-containing neurons supplying the rat digestive system: differential distribution and expression pattern.

In the enteric nervous system, calcitonin gene-related peptide (CGRP) immunoreactivity is localized to a substantial number of capsaicin-sensitive afferent fibers and to intrinsic neurons and processes. CGRP immunoreactivity detected by immunohistochemistry represents the expression of two distinct genes, the calcitonin/alpha-CGRP and the beta-CGRP genes, which have different tissue distributions. In the present study, we used (1) in situ hybridization histochemistry and ribonucleic acid (RNA) blot hybridization with RNA probes complementary to the divergent sequences of alpha- and beta-CGRP messenger RNAs (mRNAs) to differentiate which CGRP gene was expressed in enteric and afferent neurons; and (2) axonal transport approaches in combination with CGRP immunohistochemistry to define the location of CGRP-containing afferent neurons supplying the digestive system. In situ hybridization histochemistry with [35S]-labeled RNA probes indicated that in the gastrointestinal tract beta-CGRP mRNA, but not alpha-CGRP mRNA, was expressed in enteric neurons confined to the myenteric and submucous plexuses of the small and large intestine. In dorsal root and vagal sensory ganglia, mRNAs for alpha-CGRP and beta-CGRP were both present in a vast population of neurons, with an overlapping pattern, even though the alpha-CGRP signal appeared more intense. RNA blot hybridization analysis showed a single band of hybridization at 1.2 Kb with the beta-CGRP RNA probe in RNA extracts from muscle layer-myenteric plexus and submucosal layer preparations of the ileum, and from dorsal root ganglia; it also showed a single band at 1.3 Kb with the alpha-CGRP RNA probe in extracts from dorsal root ganglia, but not from the intestine. These findings further support the differential expression of alpha- and beta-CGRP mRNAs. Retrograde transport of fast blue or fluorogold coupled with CGRP immunohistochemistry demonstrated that the vast majority of CGRP-containing afferent neurons supplying the stomach, proximal duodenum, and pancreas were located in dorsal root ganglia at the middle and lower thoracic and at the upper lumbar levels, and represented a major component of the afferent innervation of these viscera (up to 89%). Approximately 50% of CGRP-immunoreactive afferent neurons also expressed tachykinin (TK) immunoreactivity, as shown by triple labeling. Only a minor component of the afferent innervation of the stomach, duodenum, and pancreas derived from vagal CGRP-containing neurons (less than 8%). A large portion of these neurons (an average of 62%) also contained TK immunoreactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

降钙素基因相关肽(CGRP)mRNA在大鼠淋巴细胞中的表达

最近,我们研究发现大鼠胸腺和淋巴结淋巴细胞中存在降钙素基因相关肽（ＣＧＲＰ）样免疫反应活性。应用人工合成的可特异扩增降钙素／降钙素基因相关肽基因部分片断的寡核苷酸引物,通过逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）,检测在大鼠脊髓背根神经节、胸腺细胞及肠系膜淋巴结淋巴细胞中是否存在ＣＧＲＰ ｍＲＮＡ,进一步研究大鼠淋巴细胞能否合成ＣＧＲＰ。结果显示,通过ＲＴ－ＰＣＲ从脊髓背根神经节（阳性对照）、胸腺和淋     

NPY-, galanin-, VIP/PHI-, CGRP- and substance P-immunoreactive neuronal subpopulations in cat autonomic and sensory ganglia and their projections

Summary The neuronal subpopulations in the cat stellate, lower lumbar and sacral sympathetic ganglia were studied with regard to the cellular distribution of immunoreactivity to tyrosine hydroxylase (TH), acetylcholinesterase (AChE) and various neuronal peptides. Coexistence of neuropeptide Y (NPY)- and galanin (GAL)-like immunoreactivity (LI) was found in a high proportion of the neuronal cell bodies; these cells also contained immunoreactivity to TH, confirming their presumably noradrenergic nature. Some TH- and GAL-immunoreactive principal ganglion cells lacked NPY-LI. Two populations (scattered and clustered) of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-positive cell bodies were found in the sympathetic ganglia studied. The scattered VIP/PHI neurons also contained AChE-LI, calcitonin gene-related peptide (CGRP)-and, following culture, substance P (SP)-LI. The clustered type only contained AChE-LI. In the submandibular and sphenopalatine ganglia, neurons were AChE- and VIP/ PHI-immunoreactive but lacked CGRP- and SP-LI. Many GAL- and occasional TH-positive neurons were found in these ganglia. In the spinal ganglia, single NPY-immunoreactive sensory neuronal cells were observed, in addition to CGRP- and SP-positive neurons. The present results show that there are at least two populations of sympathetic cholinergic neurons in the cat. Retrograde tracing experiments indicate that the scattered type of cholinergic neurons contains four vasodilator peptides (VIP, PHI, CGRP, SP) and provides an important input to sweat glands, whereas the clustered type (containing VIP and PHI) mainly innervates blood vessels in muscles.     

Pituitary adenylate cyclase activating polypeptide is expressed in autonomic neurons

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide, which is present in neuronal elements of several peripheral organs, and thus a putative neurotransmitter/modulator. In the present study, the expression of PACAP in two parasympathetic ganglia (otic, sphenopalatine) and one mixed parasympathetic/sensory ganglion (jugular-nodose) in rat was characterized by use of in situ hybridization and immunocytochemistry and compared to that of VIP and calcitonin gene-related peptide (CGRP). PACAP and VIP were expressed in virtually all nerve cell bodies in the otic and sphenopalatine ganglia; PACAP and VIP were also expressed in subpopulations of nerve cell bodies in the jugular-nodose ganglion. CGRP was expressed in numerous nerve cell bodies in the jugular-nodose ganglion and in a few, scattered, nerve cell bodies in the sphenopalatine ganglion. In the otic and sphenopalatine ganglia, PACAP- and VIP-like immunoreactivities were frequently co-localized; in the jugular-nodose ganglion, PACAP-like immunoreactivity was frequently co-localized with CGRP-like immunoreactivity in presumably sensory neurons and to a lesser extent with VIP in parasympathetic neurons. Thus, PACAP is synthesized and stored in autonomic parasympathetic neurons as well as in vagal sensory neurons, which provides an anatomical basis for the diverse effects of PACAP previously described.     

Innervation of pulmonary neuroendocrine cells and neuroepithelial bodies in developing rabbit lung.

We investigated the development of innervation of the pulmonary neuroendocrine cell (PNEC) system composed of single cells and organoid cell clusters, neuroepithelial bodies (NEB) in rabbit fetal and neonatal lungs. To visualize the nerve fibers and their contacts with PNECs/NEBs, we used confocal microscopy and multilabel immunohistochemistry (IHC) with pan-neural marker, synaptic vesicle protein 2 (SV2), and serotonin (5-HT) as markers for PNECs/NEBs, and smooth muscle actin or cytokeratin to identify airway landmarks. The numbers and distribution of PNEC/NEB at different stages of lung development (E16, 18, 21, 26, and P2) and the density of innervation were quantified. First PNECs immunoreactive for 5-HT were identified in primitive airway epithelium at E18 as single cells or as small cell clusters with or without early nerve contacts. At E21 a significant increase in the number of PNECs with formation of early innervated NEB corpuscules was observed. The overall numbers of PNECs/NEBs and the density of mucosal, submucosal, and intercorpuscular innervation increased with progressing gestation and peaked postnatally (P2). At term, the majority of NEBs and single PNECs within airway mucosa possessed neural contacts. Such an extensive and complex innervation of the PNEC system indicates a multifunctional role in developing lung and during neonatal adaptation.     

Developmentally regulated expression of the calcitonin gene related peptide (CGRP) in rat lung endocrine cells

Calcitonin (CT) and the calcitonin gene-related peptide (CGRP) are generated by alternative RNA processing from a single CT/CGRP gene. Recently, we reported the existence of CGRP-immunoreactivity and CGRP mRNA in endocrine cells or Kulchitsky (K) cells of human and rat lung [Wada et al. 1987b]. In this report, an examination was made of developmental changes in the expression of the CGRP gene in rat lungs by immunohistochemistry, radioimmunoassay (RIA) and Northern hybridization. CGRP-positive K-cells in lung tissue appeared on the 18th day of gestation. Their number was greatest on the 20th day of gestation and then decreased postnatally. The level of CGRP in rat lung was found to be highest in a 1-day-old neonate by RIA. In the Northern hybridization of rat lung using the CGRP 3' non-coding region (exon 6) of the first human CT/CGRP gene as the probe, 1.0 kilobase (kb) CGRP mRNA was found to be abundant on the 20th day of gestation and in a 1 day-old neonate. It thus appears that CGRP in rat lung is essential for pulmonary adaptation at birth and/or from the last intrauterine stage to the early neonatal period.     

Ultrastructural studies on calcitonin gene-related peptide-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia: Evidence for the colocalization of different peptides in single secretory granules

Summary Calcitonin gene-related peptide (CGRP)-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia have been studied by means of single and double immunogold labelling techniques. Peptide-immunoreactive neurones are generally B- or C-type cells of small size, with well developed rough endoplasmic reticulum and scanty neurofilaments. In neurones classifiable as A₂-type cells, i.e. larger neurones with a lighter cytoplasm due to the presence of poorly developed Nissl bodies and numerous neurofilaments, only CGRP immunoreactivity was detected. Immunolabelled structures were identified as large (60–100 nm diameter), electron-dense, membranebounded p-type granules. They were observed only in neuronal cell bodies or in the intraganglionic portions of the axons. No granules immunoreactive to the antisera applied in this study were observed in non-neuronal cells. Immunostaining experiments with different combinations of the antisera revealed, in some cells, the presence of double immunolabelled granules; in particular localization of CGRP and tachykinins, CGRP and somatostatin, and tachykinins and somatostatin to single secretory granules was demonstrated. The finding that more than one peptide is localized to the same secretory granule supports the postulate that peptides are co-released upon nerve stimulation providing morphological support for physiological and pharmacological data demonstrating an interaction between different peptides in the modulation of synaptic activity.     

Distribution of five peptides, three general neuroendocrine markers, and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: an immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I-II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I-III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I-II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.