Immunohistochemical evidence for cholecystokinin-like peptides in neuronal cell bodies of the rat spinal cord

Summary Cholecystokinin-like immunoreactivity has been demonstrated by radioimmunoassay and immunocytochemistry in the spinal cord of various mammals, in particular in nerve fibers of the superficial layers of the posterior column, but had not been detected in neuronal cell bodies. We report immunohistochemical evidence for the presence of a group of cholecystokinincontaining neuronal cell bodies in the lumbar spinal cord of the rat. This group of cells is only visualized after direct injection of colchicine into the spinal cord and is located near the central canal in the intermedio-medial nucleus of area X of Rexed.     

Localization of endothelin A receptors in the rat pituitary TSH cells: light- and electron-microscopic immunohistochemical studies

Endothelins modulate hormonal secretion in the pituitary gland. Intense signaling of endothelin A receptors (ET(A)R) has been detected by in situ hybridization, binding assay and receptor autoradiography. We used light- and electron-microscopic immunohistochemistry of ET(A)R with polyclonal antibody against a synthetic peptide corresponding to the carboxyl terminus (403-427) of human ET(A)R. Immunoreactivity was observed in 6-8% of anterior pituitary cells, which were rather large polygonal or stellate cells. These cells were often clustered. Double-staining immunofluorescence showed that the ET(A)R-positive cells immunoreacted with antibody against the beta-subunit of thyroid-stimulating hormone (TSH), but not adrenocorticotropic hormone (ACTH) or lutenizing hormone beta (LHbeta). Pre- and postembedding electron-microscopic immunohistochemistry showed that ET(A)R-positive cells had vacuolated or parallel-lined rough endoplasmic reticulum (rER) and numerous round granules in their periphery and the elongated processes. By pre-embedding immunohistochemistry, diaminobenzidine tetrahydrochloride (DAB) products were shown to be mostly located around the granules and occasionally underneath the plasma membrane. By postembedding immunohistochemistry, granules in the ET(A)R-positive cells were 90-150 nm in diameter, and colloidal gold particles due to ET(A)R were associated with about 10% of these granules. These results indicate that ET(A) receptors are associated mostly with the secretory granules of TSH cells.     

Detection of calcitonin gene expression in human infant and monkey carotid body chief cells by in situ hybridization

Calcitonin mRNA was detected in human and monkey carotid bodies by in situ hybridization histochemistry, using a ³⁵S-labeled oligonucleotide probe for human calcitonin. In both human and monkey carotid body, moderate to high hybridization signal for calcitonin mRNA was observed in all cases. The hybridization signal in the formalin-fixed, paraffin-embedded samples was comparable to that obtained from frozen paraformaldehyde-fixed tissue. Our observations extend the finding of calcitonin-like immunoreactivity in the carotid body chief cells and indicate that calcitonin is produced in the carotid body, probably in the chief cells.     

Glucagon-like peptide 1 immunoreactivity in gastroentero-pancreatic endocrine tumors: a light- and electron-microscopic study

The preproglucagon gene encodes, in addition to glucagon, two smaller peptides with structural similarity: glucagon-like peptides 1 and 2. Glucagon-like peptide 1 (GLP-1) 7–36 amide is the most powerful incretin candidate. In the present study, GLP-1 immunoreactivity was investigated in tissue specimens of various types of gastroenteropancreatic tumors, and the serum-levels of GLP-1 were assayed. Immunohistochemical staining of 88 tumors revealed GLP-1 immunoreactivity in 17 neoplasias (19.3 %), viz., in 7 out of 33 non-functioning tumors, 4 out of 20 gastrinomas, 4 out of 13 insulinomas, 1 out of 3 vasoactive-intestinal-polypeptide (VIP)omas and 1 adrenocorticotropic-hormone (ACTH)-producing tumor. In these tumors, GLP-1-immunoreactive cells were distributed either diffusely, arranged in clusters, or as single cells. All GLP-1-positive tumors were immunoreactive for glucagon or glicentin, 10 tumors were immunoreactive for pancreatic polypeptide, and 8 tumors for insulin. Ultrastructural analysis of 8 GLP-1-positive tumors, with the immunogold technique, demonstrated GLP-1 immunoreactivity mainly in cells resembling the A-cells of the pancreas or the L-cells of the gut. Of the 17 GLP-1-immunoreactive tumors, 15 were primarily located in the pancreas. Additionally, 2 non-functioning tumors of the rectum were GLP-1 immunoreactive. Five tumors were GLP-1 immunoreactive from 9 patients with multiple endocrine neoplasia I syndrome. Patients with GLP-1-immunoreactive tumors were characterized by a significantly lower rate of distant metastases (P<0.01) and a higher rate of curative resections (P<0.05). In 2 out of 22 patients, elevated serum-levels of GLP-1 were found: one patient with a vasoactive-intestinal-polypeptide (VIP)oma and 1 patient with a non-functioning tumor. This indicates that GLP-1 might be secreted at least by a few gastroenteropancreatic endocrine tumors.     

Sympathetic innervation of the carotid bifurcation in the rabbit and cat: Blood vessels,carotid body and carotid sinus

Summary Two postganglionic branches of the superior cervical ganglion enter the area of the carotid bifurcation in the rabbit and the cat. The common and external carotid arteries receive a rich adrenergic nerve supply, which can be demonstrated by fluorophores of biogenic amines appearing after formaldehyde treatment. The internal carotid artery is only sparsely innervated; however, it shows a dense sympathetic supply at the site of pressor receptors. Following removal of the superior cervical ganglion, a total loss of fluorescent adrenergic nerves occurs and degeneration of nerve endings possessing dense core vesicles is conspicuous. These nerve terminals are situated mainly subendothelially in the carotid body sinusoids; they only rarely terminate on type I cells.     

The histology of the carotid bodies in highlanders from Ladakh

A histological study was made of the carotid bodies in a boy and three adult male highlanders born and residing between altitudes of 3300 m to 4200 m in Ladakh. The carotid bodies were enlarged in two of the men, and in all four subjects showed increased numbers and enlargement of the dark variants of the chief cells of the glomic tissue. In these dark cells the cytoplasm was voluminous, formed streamers and contained many intracytoplasmic vesicles of which some had mused to form larger vesicles that appeared to have discharged from the cell surface. Immunohistochemical studies showed that these cells contained considerable amounts of the peptide met-enkephalin. It is thus considered that prominence of dark cells containing this peptide is characteristic of sustained exposure of the carotid bodies to hypobaric hypoxia. In the middle-aged highlander there was a prominence of sustentacular cells which encroached upon the cores of chief cells and this may be associated with the characteristic loss of hypoxic ventilatory response in the highlander.     

A comparative study of the distribution of carotid body type-I cells and periadventitial type-I cells in the carotid bifurcation regions of the rabbit,rat, guinea-pig and mouse

Summary The bilateral distribution of carotid body type-I cells was investigated in five rabbits, rats, guinea-pigs and mice by serially sectioning the carotid bifurcation regions. Carotid body type-I cells occurred bilaterally in close proximity to the wall of the internal carotid artery in the rabbit, rat and mouse and to the wall of the ascending pharyngeal artery in the guinea-pig. The rat carotid body was sometimes recessed into the lateral aspect of the superior cervical ganglion and was the most easily defined organ in the four animals studied. Caudally, and separate from the principal mass of carotid body type I cells, isolated groups of periadventitial type-I cells were observed in the connective tissues around the internal carotid artery and adjacent to the carotid bifurcation and common carotid artery in the rabbits only. An overall picture of the carotid body in the four animals was constructed. In all specimens rostral-caudal dimensions were recorded and compared bilaterally.The authors are indebted to Mr. Stephen Jones and Miss Alison Field of the Department of Histopathology, St Bartholomew's Hospital, for expert assistance in the preparation of the material; Miss J. McClelland and Miss C. Slatter for illustrations, and Mr. A. J. Aldrich and Mr. P.S. Hazell for photography. This work was supported by a grant from the Wellcome Trust to one of us (M. de B. D.)     

Localization of acetylcholinesterase in dissociated cell cultures of the carotid body of the rat

Summary The localization of acetylcholinesterase (AChE) was investigated at the cellular and subcellular levels in dissociated cell cultures of the carotid body of the neonatal rat, prepared by the methods of Fishman and Schaffner (1984). In the presence of iso-OMPA, which blocks non-specific cholinesterase, staining was confined almost exclusively to glomus-cell clusters and occasional isolated cells. These clusters grow as discrete islands scattered throughout the culture and display typical catecholamine (CA) fluorescence as in vivo. AChE staining was abolished or reduced by the cholinesterase inhibitors eserine (30–100 M), or (the poorly lipid soluble) echothiophate (8 (M). Processing of the same culture sequentially for the demonstration of both AChE and CA revealed that glomus-cell clusters and individual glomus cells were consistently positive for both. In electron micrographs AChE reaction product was associated intracellularly with the nuclear envelope and cytoplasm of glomus cells (identified by their characteristic dense cored granules), as well as extracellularly with the boundaries of contiguous glomus cells. Significantly, reaction product occurred in some glomus cell profiles that had both dense-cored and clear (cholinergic-like) vesicles. These findings are discussed in the context of a possible dual (adrenergic/cholinergic) function status of glomus cells in the rat's carotid body.     

Distribution of carotid body type I cells and other periadventitial type I cells in the carotid bifurcation regions of the rabbit

Summary The distribution of carotid body type I and periadventitial type I cells in the carotid bifurcation regions was investigated unilaterally in seven and bilaterally in two New Zealand White rabbits. Carotid body type I cells occurred in close proximity to the wall of the internal carotid artery immediately rostral to the carotid bifurcation, within a division of connective tissue with defineable but irregular borders. Caudally, and separate from the main mass of carotid body type I cells, isolated groups of periadventitial type I cells lay freely in the connective tissue around the internal carotid artery and alongside the carotid bifurcation and common carotid artery. A overall picture of the carotid body in the rabbit was reconstructed and the occurrence and significance of periadventitial type I cells discussed.The authors are indebted to Mr. Stephen Jones of the Department of Histopathology, St Bartholomew's Hospital, for expert assistance in the preparation of the material, and to Mr. A.J. Aldrich of the Department of Anatomy for photography. This work was supported by a grant from the Wellcome Trust to one of us (M. de B.D.)     

Immunohistochemistry of Grandry corpuscles in the oral mucosa of the duck bill: a light- and electron-microscopic study

Grandry corpuscles in the oral mucosa of the upper bill of the duck were immunohistochemically studied using antisera against calcitonin gene-related peptide (CGRP), galanin, methionine-enkephalin, neuropeptide Y (NPY), somatostatin, substance P (SP) and vasoactive intestinal peptide (VIP). Grandry corpuscles in the lamina propria selectively showed only SP-like immunoreactivity. Herbst corpuscles distributed near Grandry corpuscles were negative to all antisera applied. Although immunoreactive products in the Grandry corpuscles were found as granules in the peripheral cytoplasm of the Grandry cell, the axon terminals and satellite cells exhibited no reactivity. In pre-embedding electron-microscopic sections, SP-like immunoreactive products visualized with 3,3-diaminobezidine were localized in the granules of Grandry cells, but no labeling was observed in the cytoplasmic matrix or cell organelles. Electron-immunocytochemical labeling with colloidal gold by the post-embedding method clearly demonstrated that the SP antigen was localized only in the granules. It is presumed that Grandry cells have a secretory function. However, the function and the method of release of the SP contained in the observed granules remains obscure. Some CGRP-, NPY-, SP- and VIP-like-immunoreactive nerve fibers with varicosities associated with blood vessels and nerve fiber bundles of various sizes were observed in the lamina propria, but no such fibers penetrated into the intraepitherial layer. Nerve fibers positive for SP and VIP were also found in the interlobular connective tissue of the palatine glands. Some SP-positive neurons were detected in the vicinity of the palatine glands.     

Co-existence of gametic and agametic seminiferous tubules in a chimeric mouse. A light- and electron-microscopic study

Six chimeras, including 4 phenotypic males and 2 females, were produced by aggregation of F1 (C57BL x BALB/c) and Swiss white embryos. All were fertile, except 1 male, whose deviation in testicular structure prompted this light- and electron-microscopic study. This chimera had a well-developed sperm-conducting system, sperm in the epididymis and active accessory sex glands. The testes displayed typical parenchymal and stromal components with the important exception of co-existence of gametic and agametic seminiferous tubules. These tubules were organized in territories of quasi-lobular configurations which appeared to open separately into rete testis. The former corresponded to normally developed and active seminiferous tubules, while the latter were solid testicular cords devoid of any germ cells and embedded in solid masses of interstitial (Leydig) cells. Special mitochondrial transformations were identified in sustentacular (Sertoli) cells of both types of tubules, in maturing spermatids and sperm. These and other submicroscopic sperm defects might be the cause of infertility.     

The effect of peripheral administration of peptides on food intake, glucose and insulin in wolf pups

In the studies reported here we demonstrate that bombesin decreases food intake in wolf (Canis lupus) pups without altering glucose or insulin levels. A high dose of cholecystokinin-octapeptide (CCK, 5 μg/kg) decreased food intake. CCK produced a transient increase in insulin, without altering glucose. Glucagon (0.5 mg/kg) failed to decrease food intake despite producing a marked hyperglycemia and hyperinsulinemia. Calcitonin was ineffective at decreasing food intake, although it did decrease the time spent feeding. These studies suggest a potential role for peripheral peptides in food regulation in the wolf.     

C-PON immunoreactive neurons in the neostriatum of the hedgehog (Erinaceus europaeus): a correlated light- and electron-microscopic study

The present study provides light- and electronmicroscopic immunocytochemical data on the presence of neurons that are immunoreactive to the C-terminal flanking peptide of neuropeptide Y, C-PON, in the neostriatum of the hedgehog (Erinaceus europaeus). Positive neurons have mostly fusiform or round perikarya from which two to four poorly branched processes arise. Immunostained fibers and puncta are also evenly distributed throughout the neostriatum. Ultrastructurally, each neuron exhibits a deeply invaginated nucleus surrounded by abundant cytoplasm with a well-developed rought endoplasmic reticulum and Golgi apparatus. Positive neurons receive symmetric and asymmetric synapses from unlabeled terminals. The results of this study can be correlated with previous findings, as the C-PON-positive neurons of the hedgehog resemble medium-sized neostriatal neurons that are known to be local circuit neurons exhibiting C-PON in the rat. Thus, a high degree of C-PON neuronal system phylogenetic conservation and function can be postulated for the neostriatum of mammals.     

Calcium binding sites in the vesicles of the carotid and aortic body chief cells

Summary Chief cells of the carotid and aortic body chemoreceptors possess numerous cytoplasmic dense-core vesicles which are known to contain primarily dopamine. Following fixation in solutions containing 50 mM CaCl₂, a 20–30 nm electron-dense particle (EDP) is often observed eccentrically located in many of the vesicles. Approximately 44 % of the carotid body and 16 % of the aortic body vesicles contain an EDP. The EDP probably represents the Ca^{+ +} binding site critical to the stimulus-secretion coupling events culminating in exocytosis of these vesicles. The presence of Ca^{+ +} in the cytoplasmic vesicles was verified by electron probe X-ray microanalysis.Supported by a Grant-in-Aid from the American Heart Association (77630) and by funds contributed in part by the Texas Affiliate. The authors wish to thank Ms. Teri Heitman for her excellent technical assistance     

The occurrence and distribution of certain polypeptides within the human carotid body

Summary Both carotid bodies from 26 patients coming to necropsy were fixed in 10% neutral buffered formalin and sections 4 m thick were stained for various peptides by use of the immunogold technique. The results show that the human carotid body contains met- and leu-enkephalin, substance P, vasoactive intestinal peptide (VIP), neurotensin and bombesin. The distribution of these six peptides within the carotid body differs. Thus met- and leu-enkephalin are both present predominantly within glomic chief cells but with a marked tendency to favour the dark variant of these cells. Substance P and VIP both show a weak immunoreactivity in comparison to the enkephalins and are present in all three variants of chief cell. Neurotensin shows the weakest immunoreactivity of all and is restricted to a few glomic chief cells in a minority of cases. Bombesin also shows a weak immunoreactivity in glomic chief cells but a strong reaction in glomic arteries and arterioles. In these vessels bombesin appears to be confined to smooth muscle cells in the media but we cannot say whether it is secreted by them or merely bound to receptor sites on their membranes. These findings are related to quantitative data on the concentration of peptides in the human carotid body from a previous paper with which we were associated.     

Gonadal development and sex differentiation in the cichlid fish Cichlasoma dimerus (Teleostei, Perciformes): a light- and electron-microscopic study

Although the overall pattern and timing of gonadal sex differentiation have been established in a considerable number of teleosts, the ultrastructure of early stages of gonadal development is not well documented. In this study, gonads from larval and juvenile stages of laboratory-reared Cichlasoma dimerus were examined at the light-microscopic and ultrastructural levels. This freshwater species adapts easily to captivity and spawns with high frequency during 8 months of the year, providing an appropriate model for developmental studies. Larvae and juveniles were kept at a water temperature of 26.5 +/- 1 degrees C and a 12:12 hour photoperiod. Gonadal development was documented from 14-100 days postfertilization, covering the period of histologically discernible sex differentiation. Gonadal tissue was processed according to standard techniques for light and electron microscopy. C. dimerus, a perciform teleost, is classified as a differentiated gonochorist, in which an indifferent gonad develops directly into a testis or ovary. On day 14, the gonadal primordium consists of a few germ cells surrounded by enveloping somatic cells. Ovarian differentiation precedes testicular differentiation, as usual in teleost fishes. The earliest signs of differentiation, detected from day 42 onward, include the onset of meiotic activity in newly formed oocytes, which is soon accompanied by increased oogonial mitotic proliferation and the somatic reorganization of the presumptive ovary. The ovarian cavity is completely formed by day 65. Numerous follicles containing perinucleolar oocytes are observed by day 100. In contrast, signs of morphological differentiation in the presumptive testis are not observed until day 72. By day 100, the unrestricted lobular organization of the testis is evident. The latest stage of spermatogenesis observed by this time of testicular development is spermatocyte II.     

Gastrin/cholecystokinin-like post-prandial variations: quantitative and qualitative changes in the haemolymph of penaeids (Crustacea Decapoda)

Summary Gastrin/cholecystokinin-like (G/CCK) peptides from the haemolymph of two penaeidsPenaeus japonicus andP. stylirostris (Crustacea, Decapoda) were characterized by gel filtration. In starved shrimps, four classes of peptides were found. One fraction elutes in the void volume, a second has a molecular weight around 5 kDa, a third between 1 and 2.5 kDa, and the fourth fraction is composed of very small peptides. Differences exist between the two species concerning the relative proportion of each molecule.After a meal, there is a two-fold increase of total G/CCK-like peptides and marked qualitative changes are described. Briefly, high molecular weight fractions decrease while low molecular ones increase. After 5 h a return to the initial concentrations is observed. These changes are more pronounced inP. stylirostris. Such variations suggests a role for these peptides in regulation of crustacean digestive processes.