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The pelvic flexure portion of the equine large colon is the proposed location of a pacemaker mechanism. This study was conducted to ascertain whether the distribution of certain putative neurotransmitters differs at the pelvic flexure compared to other sampling sites. Tissue samples were collected from the intestinal tracts of six horses. Serial sections from these samples were reacted with primary antisera specific for substance P, vasoactive intestinal polypeptide (VIP), methionine-Enkephalin, and calcitonin gene-related peptide (CGRP). The regional distribution of immunoreactive neuronal elements was uniform for each of the neuropeptides except VIP. Although neurons exhibiting VIP-like immunoreactivity were abundant throughout the colon, they were somewhat more plentiful near the apex of the pelvic flexure and the left dorsal colon. These neurons may participate in the initiation and propagation of the propulsive/retropulsive contraction waves, which emanate from this location and are believed to lend a sphincter-like capacity to the pelvic flexure. The submucosal plexus was replete with neurons with intense substance P and VIP-like reactivity. Reactive fibers left submucosal ganglia to project to the intestinal mucosa, reflecting a possible secretogogic role for these neurons. This role may be especially important for the horse as a hindgut fermenter. There were abundant methionine-Enkephalin and substance P-like reactive varicosities throughout the myenteric plexus, many of which established a pericellular plexus of varicose fibers. The abundance of these varicosities, which may correlate with a high degree of neuronal integration, did not vary regionally. These data may enhance our understanding of both normal colonic peristalsis and motility disorders caused by a depletion of these neuropeptides.
Anat Rec 1993 Jun
PMID:Neuropeptide distributions in the colon, cecum, and jejunum of the horse. 768 32

We describe, for the first time, the development of a technique for a long-term selective culture of endocrine (PE) cells from the lungs of normal animals. Epithelial cells were isolated from 1-day-old hamster lungs through mechanical and enzymatic dissociation with collagenase type II. Cells were then cultured in HITES medium which contained RPMI 1640, hydrocortisone, insulin, transferrin, estradiol, sodium selenite, and supplemented with 5% fetal bovine serum (FBS), or medium which contained HITES medium supplemented with bovine serum albumin, phosphoethanolamine, arginine vasopressin, bombesin, and 2% FBS (9N). HITES medium, originally developed for establishment and long-term culture of human small cell lung cancer (SCLC) cell lines, allowed propagation of normal hamster PE cells up to 12 months as a mixed floating-attached cell culture. No difference was noted in the results using HITES or 9N. By 3 months, 80% of the cultured cells contained characteristic dense-core (endocrine type) granules. The cultured PE cells also expressed creatine kinase brain isoenzyme, and general NE markers including neuron specific enolase, and amine handling enzyme activity within the range of SCLC cell lines. Moreover, cultured PE cells contained and secreted immunoreactive calcitonin (iCT) which had a molecular profile similar to that of intact hamster lung. This long-term culture technique should markedly assist in elucidating the role of PE cells in health and disease.
Anat Rec 1993 May
PMID:Long-term selective culture of hamster pulmonary endocrine cells. 838 31

Despite four decades of investigation, the function of pulmonary neuroendocrine cells (NEC) remains unclear. Since NEC secretory products may influence airway growth or differentiation or alter airway smooth muscle tone, increased numbers of NEC seen in bronchopulmonary dysplasia (BPD) may be partially responsible for the genesis of the structural and pathophysiological alterations seen in this disease state. Changes in airway structure were studied in six infants dying with BPD and six conceptional age-matched control infants dying of noncardiopulmonary disease. Changes in bombesin-, calcitonin-, and serotonin-immunoreactive NEC were quantified in lung specimens from three infants who died at 2 months of age with severe BPD and three conceptional age-matched controls. There were no differences in either bronchiolar or bronchial airway epithelial areas, but significant increases in bronchiolar (1.8-fold) (P < 0.001) and especially bronchial smooth muscle (2.5-fold) (P < 0.001) were documented in infants with BPD. Few bombesin-, calcitonin-, and serotonin-immunoreactive cells were identified in cartilaginous airways; however, there was a clear increase in the total number of bronchiolar immunoreactive cells in infants with severe BPD (28.5 +/- 11.2 cells/mm airway epithelium) compared to control infants (4.5 +/- 4.9) (P < 0.05). Our results confirm that airway wall composition does change in BPD, but there is either no or an inverse correlation between NEC number and airway epithelial and smooth muscle areas and cell numbers. The role of NEC secretory products in airway smooth muscle growth and function requires further investigation.
Anat Rec 1993 May
PMID:Pulmonary neuroendocrine cells in pediatric lung disease: alterations in airway structure in infants with bronchopulmonary dysplasia. 850 96

Dispersed newborn hamster lung cells were established in vitro in a defined, low-serum growth medium. Neuroendocrine markers (immunohistochemistry for bombesin/gastrin-releasing peptide and calcitonin) revealed a cellular predominance of pulmonary neuroendocrine (PNE) cells. While the supernatant concentration remained stable, the concentration of PNE cell immunoreactive calcitonin (iCT) gradually declined over 4 weeks. Supplementation of the medium with nicotine for 3 weeks prevented this decline in cellular iCT. Concurrently, the number of cells and [3H]thymidine incorporation were significantly increased. The stimulatory effect of chronic nicotine was reversed by the coadministration of the nicotinic antagonist hexamethonium. In another set of experiments, prior multiple transplacental nicotine pretreatments resulted in a significant increase in iCT in the lungs of newborns; when these lungs were subsequently placed in cell culture without nicotine, despite the higher concentration of iCT, there was a drop in iCT similar to that observed in the control culture. In contrast, in vivo, the lung iCT remained significantly elevated at 1 week post-parturition. Cell culture supernatants were analyzed at week 4 for the evoked release of iCT; cholinergic-nicotinic agonists promptly increased the supernatant iCT, which was blocked by nicotinic but not by muscarinic antagonists. We suggest that this in vitro system provides a useful tool to study directly the PNE cell. The acute and chronic effects of nicotine are most likely related to stimulation of cholinergic-nicotinic receptors on iCT-containing PNE cells.
Anat Rec 1993 May
PMID:Cholinergic-nicotinic control of growth and secretion of cultured pulmonary neuroendocrine cells. 850 98

This study investigated the colocalization of the peptide hormones bombesin or calcitonin with calcitonin gene related peptide (CGRP) in neuroendocrine cells (NE) in the lungs of human fetuses of varying gestational ages and in the lungs of newborn infants who died with acute or chronic lung disease in the first weeks or months after birth. Double immunolabeling of dense core granules for these peptides was also studied in this same patient population. On-grid double gold immunolabeling was carried out on 29 subjects using anti-bombesin and anti-CGRP and on 22 subjects using anti-calcitonin and anti-CGRP as primary antibodies, the secondary antibodies being labeled with different-size gold spheres. Colocalization of both bombesin and calcitonin with CGRP was demonstrated, not only in the same NE cell, but also on the same dense core granule. Colocalization was rarely found in normal fetuses, and most frequently found in newborn infants with acute lung disease, usually hyaline membrane disease (HMD), or with the development of chronic lung disease in the first weeks or months after birth. Double labeling of the same dense core granules might imply action of peptides in concert, or perhaps one peptide acting in a paracrine role (e.g., on bronchial or bronchiolar smooth muscle) and the second peptide acting in an autocrine fashion on the parent cell (e.g., in the regulation of granule production or release).
Anat Rec 1993 May
PMID:Colocalization of peptide hormones in neuroendocrine cells of human fetal and newborn lungs: an electron microscopic study. 850 8

Knowing that small-granule endocrine cells develop in organ cultured fetal lungs, we investigated whether the cells produce regulatory peptides in vitro, and if sufficient amounts appear to permit using the cultures as an experimental system for physiological study of secretory mechanisms. The paired lungs from 14-day and 15-day fetal rats were organ cultured for 1-8 days and examined daily for development of immunoreactivity against marker proteins and regulatory peptides associated with small-granule endocrine cells and nerves. They proved reactive against protein gene product 9.5 (PGP) and calcitonin-gene related peptide (CGRP) but not against calcitonin or neurofilament protein 200 K, although positive controls were obtained for these substances in lungs from postnatal animals. Initially PGP-like immunoreactivity is associated with cell bodies and processes of neuroblasts which run medial to the bronchial axis on day 14 and are increasingly prevalent on day 15. In 15-day explants PGP becomes detectable after a day in vitro in rare "clear cell" precursors of small-granule cells located in the epithelium lining proximate parts of the lungs, although in 14-day explants comparable reactivity is not seen until the third day (14 + 3 days). In culture PGP-positive neuroblasts increase in number, and nerve processes gradually extend down the airway to encircle the sleeve of smooth muscle that develops as the bronchial tree expands. Concurrently, the initially small clusters of small-granule cells increase in size, and new ones appear in the airway lining. By 15 + 5 days they extend to the boundary between a taller, more proximal epithelium and a glycogen-rich cuboidal layer that lines one or two most-distal generations of branches. Thereafter, the trachea and central, cartilage-bound segments of the primary bronchi mainly contain solitary endocrine cells and the more peripheral lung a mixture of single cells and clusters, much as in near-term lungs in vivo. At this stage PGP-positive nerves extend as far as the entrances of the terminal sacs, and most are distributed to the airway muscle plexus. Exceptionally, they may innervate a small-granule cell cluster, converting it into a neuroepithelial body. CGRP-like immunoreactivity initially appears in small-granule cells of 15 + 2-day cultures but does not develop in ganglion cells or nerves. It localizes to endocrine cells at all conducting airway levels, increasing in staining intensity and accounting for most if not all of the PGP-positive population between 15 + 4 - 15 + 8 days.(ABSTRACT TRUNCATED AT 400 WORDS)
Anat Rec 1993 May
PMID:Development of PGP 9.5- and calcitonin gene-related peptide-like immunoreactivity in organ cultured fetal rat lungs. 850 9

Small-granule endocrine cells differentiate in airway epithelium of intact and cultured fetal rat lungs. We noted that the cells store calcitonin gene-related peptide (CGRP) in vitro as well as in vivo and used the ionophore A23187 to test the effects of calcium on peptide secretion in this system. Lungs of 14-day and 15-day fetal rats, organ cultured for 6-9 days, were divided into groups of 5 explants each and incubated for 15 min at 37 degrees C in the basic medium containing 0 mM, 1 mM, or 10 mM CaCl2, with or without 8 microM A23187, or 10 mM EGTA. Intracellular CGRP in these explants was quantified by supraoptimal dilution peroxidase immunocytochemistry (Springall et al.: J. Pathol. 155:259-267, 1988): counts were made of endocrine cells stained with a 1/60,000 dilution of anti-CGRP and repeated on the same sections after restaining with antibody diluted at 1/1,000. Results, analyzed by Chi-square test, were expressed as % cells stained with antibody at 1/60,000 vs. those stained at 1/1,000. Immunoreactivity for CGRP was significantly reduced by A23187 in the presence of high extracellular Ca2+ (10 mM), the inference being that these cells secrete peptide hormones in response to Ca2+ influx across the plasma membrane. The organ cultures evidently can be used to assess certain physiological responses of lung endocrine cells in an accessible, relatively organotypical setting.
Anat Rec 1993 May
PMID:Calcium and ionophore A23187 lower calcitonin gene-related peptide-like immunoreactivity in endocrine cells of organ cultured fetal rat lungs. 850 10

Hamsters were exposed to 60% hyperoxia for 1 week, 3 weeks, or 3 months. The exposed animals gradually failed to gain body weight as controls. The pulmonary neuroendocrine (PNE) cell peptides, mammalian bombesin (MB) and immunoreactive calcitonin (iCT), were determined in the lung and the serum. At 1 week and 3 weeks, lung MB was unchanged while the iCT levels were markedly depleted. In contrast, the lung levels of both MB and iCT were significantly elevated at 3 months. Serum levels of MB showed an initial decline at 1 week, which was followed by augmented levels at 3 weeks and at 3 months. In contrast, serum iCT showed considerable depletion at 1 week, and also at 3 weeks, followed by increased levels at 3 months. Thus, chronic exposure to hyperoxia causes profound perturbation of PNE cell peptides. In particular, the early depletion of lung and serum iCT appears to be a unique feature of the response to hyperoxia. The principal difference between the MB and the iCT responses was the lack of an initial depletion of lung MB, and the earlier rise of serum MB to supranormal levels. It seems likely that the early peptide effects of hyperoxia are related to oxygen toxicity upon the PNE cells, while the changes noted at 3 months reflect a hyperplastic accommodation of PNE cells to the prolonged oxygen exposure with resultant increases in MB and iCT. This response is distinctly different from that we have seen previously in hamsters exposed to hyperoxia combined with a nitrosamine, or a nitrosamine alone.
Anat Rec 1993 May
PMID:Chronic hyperoxia and hamster pulmonary neuroendocrine cell bombesin and calcitonin. 850 12

Various acute stimuli, including cigarette smoke, induce hypercalcitonemia in man and hamsters. We have shown that this occurs also in thyroidectomized subjects. In the present study we have further explored this phenomenon of secretion from the lungs by studying, simultaneously, the HPLC characteristics of pulmonary tissue and serum in control hamsters and in animals immediately following short-term exposure to cigarette smoke. In addition, we have studied the immunoheterogeneity of lung calcitonin 24 hours following the acute exposure. Control lungs contained monomeric immunoreactive calcitonin (M-iCT), high molecular mass iCT (H-iCT), and CT fragments. Immediately following smoke exposure, there was an acute decrease of lung iCT by radioimmunoassay (RIA) which consisted primarily of a decrease in M-iCT by HPLC. Simultaneously, the iCT increase in the serum by RIA was shown by HPLC to involve M-iCT. Twenty-four hours after smoke inhalation, the lung iCT by RIA and M-iCT by HPLC had returned towards control levels. These findings document the molecular characteristics of lung iCT following acute cigarette smoke stimulation, and suggest that under certain circumstances M-iCT may be actively secreted by the lung. It remains to be determined whether this type of secretion reflects hemocrine or paracrine release and what the physiological role for such a secretion may be.
Anat Rec 1993 May
PMID:Heterogeneity studies of hamster calcitonin following acute exposure to cigarette smoke: evidence for monomeric secretion. 850 14

Only scant information is available in the scientific literature on the parathyroids and ultimobranchial bodies in the primitive mammals, the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus). The major aim of this paper is to describe the morphology of the monotreme parathyroid gland and to compare it with parathyroids in mammals and reptiles. The gross anatomy and light microscopic structure of the ultimobranchial body, thymus, and thyroid are also given. Animals were dissected and routine light and electron microscopic techniques used to examine the microscopic morphology. The locations of parathyroid hormone, calcitonin and calcitonin gene-related peptide in tissue sections were identified by immunostaining. Monotremes have one pair of parathyroid glands located in the thorax and they are often associated with thymic tissue but never with the thyroid which is also present in the mediastinum. Ultimobranchial bodies are ventrolateral to the commencement of the trachea. Thymic lobules with Hassall's corpuscles are scattered in the fibrofatty tissue of the mediastinum and the ventral surface of the pericardium. Histologically, principal cells, water-clear cells, and non-secretory cells were identified in the parathyroid glands. Principal cells showed polarity and had microlamellar projections that formed intercellular canaliculi. Non-secretory cells had features similar to those of thymic epithelial reticular cells. Immunostaining of parathyroid hormone showed a diffuse distribution in parathyroid principal cells and none in ultimobranchial bodies. Identification of the ultimobranchial bodies was confirmed by immunostaining. The monotreme parathyroid gland, ultimobranchial bodies and thyroid show reptilian as well as mammalian features.
Anat Rec 1999 02 01
PMID:Parathyroids and ultimobranchial bodies in monotremes. 997 12


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