Acute stimulation of chromaffin cell proliferation in the adult rat adrenal medulla.

Susceptible strains of rats develop adrenal medullary hyperplasia and neoplasia after long-term administration of the antihypertensive drug reserpine, or of other pharmacologic agents which alter neuroendocrine function. These proliferative lesions are of potential medical importance as a model for familial multiple endocrine neoplasia syndromes, and are of fundamental interest because they might elucidate mechanisms regulating chromaffin cell proliferation during normal development. To study the initiation of the adrenal lesions, chromaffin cell mitoses were counted in adult male rats injected with reserpine or control solvent for 5 days, with the final injection containing colcemid to arrest cells in mitosis. Rare mitoses were observed in mature-appearing epinephrine and norepinephrine cells in control adrenals. Reserpine caused an 8-fold increase in chromaffin cell mitoses in otherwise histologically normal glands, and the mitotic cells after reserpine administration showed marked granule depletion. Reserpine directly depletes catecholamine stores and reflexively increases neurogenic stimulation of chromaffin cells to increase catecholamine synthesis. The findings suggest that signals regulating function also regulate proliferation of mature chromaffin cells, and that prolongation of these signals or superimposed abnormalities may lead to pathologic proliferative states. The reserpine model may be a useful system for elucidating normal and pathologic mechanisms of signal transduction.

Anti-lymphocyte antibody Leu-7 (HNK-1) recognizes a constituent of neuroendocrine granule matrix.

Anti-lymphocyte monoclonal antibody HNK-1 (Leu-7) reacts with the cell surfaces of natural killer (NK) lymphocytes and with myelin-associated glycoprotein (MAG). This antibody reacts intensely with normal and neoplastic adrenal medullary cells. A small proportion of normal pancreatic islet cells, anterior pituitary, and gastroenteropancreatic endocrine cells also show Leu-7 immunoreactivity. In adrenal medulla, ultrastructural immunocytochemical studies and immunoblot analyses reveal that Leu-7 reacts with an intracellular protein of MW 75 KD which is localized within the matrices of the chromaffin granules. The MW of this protein differs from those of MAG and chromogranin A. The findings suggest that Leu-7 immunoreactivity might be a new marker for specific subsets of secretory granules.

Establishment of a continuous somatostatin-producing line of medullary thyroid carcinoma cells from BALB/c mice.

A continuous line of somatostatin-producing medullary thyroid carcinoma cells was established from a transplantable tumour in BALB/c mice. Virtually all of the somatostatin immunoreactivity co-chromatographed with somatostatin 14. The tumour cells replicated in spinner cultures with a doubling time of approximately 4 days, and the concentration of somatostatin released into the culture medium increased in proportion to the number of cells. Two- to threefold increases in amounts of stored and released somatostatin were observed after treatment of the cells with bromodeoxyuridine. This cell line might be valuable for studies of somatostatin regulation in normal and neoplastic C-cells, and for other studies of C-cell biology which require a mouse model.

Duodenal carcinoids in patients with and without neurofibromatosis. A comparative study.

Nine duodenal carcinoids from patients with von Recklinghausen's neurofibromatosis (VRNF) were investigated for their morphologic, immunocytochemical, and ultrastructural characteristics, and were compared with seven similar tumors from patients without VRNF. Strong similarities were found between tumors in each group. Irrespective of their association with VRNF, duodenal carcinoids arose in adults and usually produced jaundice, upper intestinal bleeding, or obstruction. Tumors larger than 2.0 cm had already metastasized when first detected. All tumors showed a mixed architectural pattern; five tumors associated with VRNF were of the psammomatous type, as opposed to two of those without VRNF. While no tumors showed argentaffinity, stray argyrophil cells were present only in the three tumors not associated with VRNF. All of the tumors showed immunocytochemical evidence of somatostatinomas, and only one VRNF-associated tumor showed immunoreactivity for an additional regulatory substance, as opposed to three of those not associated with VRNF. Thus, while VRNF-associated duodenal carcinoids are not otherwise distinctive, they tend to be pure somatostatinomas (eight of nine cases), whereas similar tumors unassociated with VRNF are frequently multihormonal (three of seven cases). While many more duodenal carcinoids need to be investigated systematically for their immunocytochemical profile, detection of a pure somatostatinoma in the duodenum should alert one to the possibility of coexistent VRNF.

Spontaneous proliferative lesions of the adrenal medulla in aging Long-Evans rats. Comparison to PC12 cells, small granule-containing cells, and human adrenal medullary hyperplasia.

Aging rats of the Long-Evans strain spontaneously develop diffuse and nodular hyperplasia of the adrenal medulla in association with other abnormalities commonly encountered in human multiple endocrine neoplasia syndromes. The cells which comprise the adrenal nodules resemble those in the parent tumor of the rat PC12 pheochromocytoma cell line in that they show varying degrees of spontaneous or nerve growth factor-induced neurite outgrowth in culture and they contain little or no epinephrine. In addition, cells from at least some of the nodules contain immunoreactive neurotensin and neuropeptide-Y, which are also found in PC12 cells. There are a number of striking resemblances between the cells in adrenal nodules and the small granule-containing cells in the normal rodent adrenal. The findings suggest that spontaneous rat adrenal medullary nodules and PC12 cells might be derived from small granule-containing cells, or that cells within the nodules might regain properties of immature chromaffin cells and acquire characteristics of small granule-containing cells and of PC12 cells in the course of neoplastic progression. They further suggest a possible relationship between proliferative capacity and neurotransmitter phenotype in the adult rat adrenal medulla. By virtue of their sparse epinephrine content and their small granules, the cells in adrenal medullary nodules of Long-Evans rats differ from those in adrenal medullary nodules of humans with multiple endocrine neoplasia syndromes.

Psammomatous somatostatinomas of the duodenum.

The presence of psammoma bodies in carcinoid tumors of the gastrointestinal tract is a rare occurrence; it has also been reported to be associated with features of somatostatin production by the tumor cells. The morphologic features of three such tumors arising in the duodenum were studied by a combination of histochemical, immunocytochemical, and ultrastructural techniques in an effort to delineate their secretory profile and further subclassify them. All tumors showed a mixed architectural pattern with prominent areas of glandular differentiation. The psammoma bodies were almost exclusively located within the glandular lumina. In each instance, the majority of tumor cells showed histochemical and immunocytochemical features of somatostatin-containing cells, and one tumor studied ultrastructurally showed numerous large- and small-sized intracytoplasmic secretory granules, both of which contained somatostatin. In contrast to other endocrine tumors of the duodenum that frequently have a multihormonal secretory profile, psammomatous duodenal carcinoids are associated with the exclusive presence of somatostatin within tumor cells. While many more of such examples of this uncommon tumor need to be systematically investigated for their immunocytochemical and ultrastructural characteristics, duodenal somatostatinomas need to be included in the differential diagnosis of psammomatous tumors.

Pituitary proliferative lesions in aging male Long-Evans rats. A model of mixed multiple endocrine neoplasia syndrome.

The incidence of spontaneous pituitary nodules in rats increases progressively with age. In a colony of male Long-Evans rats, 50 per cent of the 2- to 3-year-old animals harbored pituitary nodules which were composed almost exclusively of sparsely granulated prolactin (PRL) cells. Sixty per cent of the PRL nodules were multicentric and in most instances were associated with diffuse hyperplasia of PRL cells adjacent to the nodules. A progressive increase with age in the population of PRL cells was also observed. These findings suggest that the pituitary nodules may have originated from foci of PRL hyperplasia. Furthermore, five of these glands contained additional nodules of other hormonal cell types, including mixed PRL/gonadotropins (two animals), mixed PRL/thyrotropin-stimulating hormone (one), gonadotropins (one), and adrenocorticotropic hormone (one). These animals also demonstrated a high incidence of thyroidal C cell nodular hyperplasia and/or medullary thyroid carcinoma (42 per cent), adrenal medullar nodules (31 per cent), and parathyroid nodular hyperplasia (30 per cent). In human kindreds with multiple endocrine neoplasia syndromes, concomitant thyroidal C cell and adrenal medullary proliferative lesions, as well as parathyroid abnormalities, are found in type II phenotype patients, whereas pituitary, pancreatic islet, and parathyroid abnormalities develop in patients with type I phenotype. The Long-Evans rat strain, with concomitant proliferative lesions in these four endocrine organs, may provide a useful model system of a mixed multiple endocrine neoplasia syndrome and, also, of prolactin-secreting pituitary adenomas.

Long-term effects of dexamethasone and nerve growth factor on adrenal medullary cells cultured from young adult rats.

Normal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2% of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.

Spontaneous neurite outgrowth and vasoactive intestinal peptide-like immunoreactivity of cultures of human paraganglioma cells from the glomus jugulare.

The chief cells of paraganglionic tissues have morphological and functional similarities to adrenal chromaffin cells, and both cell types are derived from the neural crest. In the present investigation cells from two glomus jugulare paragangliomas were studied in culture. Approximately 50% of the cells from one tumor, and 7% from the other spontaneously formed neurite-like processes. Numerous granular and agranular synaptic-like vesicles also appeared in the process-forming cells. In contrast to findings with normal and neoplastic adrenal chromaffin cells, addition of nerve growth factor (NGF) to the culture medium had no major effects on proportion of cells with processes. Dexamethasone caused only a small decrease in process length. Culturing of the tumors also appeared to promote production of material with VIP-like immunoreactivity. It is concluded that the phenotype of paraganglioma as well as pheochromocytoma cells may be altered in vitro. Responsiveness to specific factors such as NGF or steroids, however, may vary for related tumor cell types in different anatomic locations.

C-cell hyperplasia and medullary thyroid carcinoma in the rat. An immunohistochemical and ultrastructural analysis.

Medullary thyroid carcinoma (MTC) is a distinctive neoplasm which is derived from the calcitonin-producing intrathyroidal C-cell system and which develops commonly in untreated rats of various strains. Thyroid glands of Long-Evans rats ranging in age from 3 months to 3 years showed a spectrum of C-cell proliferative abnormalities. As compared to 3-month-old control rats, thyroids from 9- to 12-month-old animals exhibited mild diffuse C-cell hyperplasia (CCH). Thyroids from animals ranging from 1 to 3 years of age exhibited progressively more severe C-cell abnormalities including severe diffuse CCH, nodular CCH, and/or MTC. In contrast to the normal basal serum calcitonin levels in controls and in animals with mild diffuse CCH, animals with severe diffuse CCH, nodular CCH, or MTC had elevated basal serum calcitonin values. Nodular CCH was characterized by the replacement and enlargement of individual follicles by C-cells. Larger foci of nodular CCH were characterized by similar changes in multiple adjacent follicles or by an irregular expansion of individual follicles. MTC was characterized by penetration of the follicular basal lamina by C-cells with extension into the adjacent thyroid stroma. In addition to the high incidence of thyroidal C-cell abnormalities, diffuse and/or nodular parathyroid hyperplasia was commonly found. There was no evidence of chronic renal failure in these animals, and the serum calcium levels were within normal limits. Although the stimulus for the initial C-cell proliferation remains unknown, the appearance of MTC is preceded by relatively prolonged phases of CCH. These findings are essentially identical with those noted in human familial MTC and indicate that the rat provides a useful model system for studying the regulation of C-cell proliferation during the processes of neoplastic development and progression.

C-cell hyperplasia. An ultrastructural analysis.

The C-cell is the source of the hypocalcemic polypeptide hormone, calcitonin. Sequential increments in calcitonin response to provocative calcium and pentagastrin infusions have been used to identify family members at high risk for the development of medullary thyroid carcinoma, a neoplasm of C-cell origin. Correlative light microscopic and immunohistochemical studies have permitted the identification of a spectrum of C-cell proliferative abnormalities ranging from C-cell hyperplasia (CCH) to invasive medullary thyroid carcinoma. Ultrastructurally, both normal and hyperplastic C-cells occupied an intrafolliculat localization, being separated from the interstitium by the follicular basal lamina and from the luminal colloid by extensions of the follicular cell cytoplasm. These relationships were maintained in areas of advanced hyperplasia where C-cells often completely encircled and displaced the follicular epithelium. Nodular CCH was characterized by the complete obliteration of the follicular space by C-cells. In these areas, prominent reduplications as well as occasional defects in the basal lamina were noted. Some of the solid C-cell nodules adjacent to areas of medullary thyroid carcinoma appeared to form by gradual replacement of follicles by C-cells and did not represent sites of intrathyroidal vascular or lymphatic extension. Two major cell types were noted in cases of CCH. One cell type which was filled with secretory granules measureing 280 nm. in diameter (type I) predominated in areas of diffuse CCH and was also found in control thyroid glands. The larger number of secretory granules in this cell type together with the relative lack of development of granular endoplasmic reticulum and Golgi regions suggested that these cells were in the storage phase of their secretory cycle. The second cell type had fewer secretory granules, which measured 130 nm. in diameter (type II), and was characteristically located in areas of nodular CCH. Type II granules were found in cells with cytologic evidence of active protein synthesis and secretion. Variations in granule morphology and cell ultrastructure may be correlated with functional variations in C-cell populations.