Characterisation of N-terminal chromogranin A and chromogranin B in mammals by region-specific radioimmunoassays and chromatographic separation methods

Chromogranin A (CgA) and chromogranin B (CgB) are acidic proteins stored in and released from hormone granules in endocrine and neuroendocrine tissue. The chromogranins are postulated to serve as pro-hormones to generate biologically active peptides, which may influence hormonal release and vascular functions or have antibacterial functions. Although N-terminal and C-terminal regions show some species amino acid homology, the chromogranins as a whole display considerable interspecies differences, which prevents their use in comparative studies of biological functions. We present four new radioimmunoassays for the measurement of defined N-terminal regions of CgA and CgB. A new radioimmunoassay for measurement of intact bovine CgA has also been developed. With these assays and two previously published ones, we have compared the cross-reactivity of chromogranins from man, cattle, sheep, goat, pig and horse and compared adrenomedullar content and serum levels of CgA from these species. We have also studied the influence of peptide concentrations and the ionic strength of the mobile phase on molecular weight estimations. Assays with antibodies directed against the N-terminal parts of CgA and CgB showed sufficient interspecies cross-reactivity to allow comparative quantification of the circulating levels in man, cattle, sheep, goat, pig and horse. Assays measuring the intact human or bovine CgA were not suitable for comparative purposes in samples from sheep, goat, pig and horse. Molecular interactions between vasostatin immunoreactive material and intact bovine CgA were demonstrated in gel permeation studies, suggesting that conclusions about the degree of N-terminal processing from elution profiles should be made with caution. Reliable interspecies comparison of chromogranins is difficult, but measurements with region-specific assays may be helpful to study concentrations of chromogranins and chromogranin-related peptides.

Molecular organization of prolactin granules. II. Characterization of glycosaminoglycans and glycoproteins of the bovine prolactin matrix.

Prolactin (PRL) granules can be isolated from the anterior pituitary gland of adult cows in nearly 50% yield by use of a procedure previously developed for the fractionation of the rat pituitary. Treatment of the isolated bovine granules with 0.2% Lubrol PX results in the solubilization of most membranes present in the fractin but has only a limited effect on the matrices, which remain aggregated and can be recovered and purified by gradient centrifugation. These membraneless PRL granules, studied in detail by morphological and biochemical techniques, were found to contain only small amounts of contaminants (primarily growth hormone granules and small membrane fragments). SDS polyacrylamide gel electrophoresis revealed that, in comparison with other fractions isolated from the bovine pituitary, the membraneless granules have a simpler polypeptide composition including PRL (approximately 85%), growth hormone (approximately 8%), as well as approximately 13 minor bands with apparent mol wt ranging from 80,000 go 45,000. Many of these minor bands are accounted for by glycoproteins, as revealed by their binding of 125I-concanavalin A, and two of these are also stained blue by the stains-all procedure, a reaction specific for acidic glycoconjugates. Chemical analyses of the membraneless granule fractin revealed the presence of a heterogeneous mixture of complex carbohydrates. Among glycosaminoglycans, the major component is heparan sulfate, while hyaluronic acid and chondroitin sulfate ar present in smaller amounts. Moreover, some of the glycoproteins are sulfated and account for over 50% of the nondialyzable 35S radioactivity found in the fraction isolated from labeled slices. Although the concentration of glycosaminoglycans and glycoproteins is relatively low in membraneless granules, the possibility that their presence in the fraction is largely due to cross-contamination and/or artifactual adsorption could be excluded on two grounds. These are: (a) electron microscope radiautography of preparations obtained from [35S]sulfate- and D-[6-3H]glucosamine-labeled slices showed a significant labeling of PRL granules in both intact cells and membraneless granule pellets, and (b) a mixing experiment showed that membraneless granules contain very little macromolecular sulfate radiactivity adsorbed from the soluble glycoconjugates present in the pituitary homogenate.

THE SIZE OF GROWTH HORMONE GRANULES IN PITUITARY ADENOMAS PRODUCING ACROMEGALY

ABSTRACT Measurements of the diameters of large numbers of granules in electron micrographs of pituitary adenomas in cases of acromegaly do not show that a typical size exists which allows of the retrospective diagnosis of the hormone secreted by the tumour. The granule diameters found in single cases show a normal distribution. The average values determined in 19 unselected cases of acromegaly range between 160 and 342 nm. They are also normally distributed about the mean for the entire group which is 238 nm. With histo-immunological electron microscopic techniques hGH can be demonstrated in small, medium and large granules. The size of hormone granules is determined by functional factors such as the speed of hormone synthesis and hormone release but not by the type of hormone secreted.

Peptides with NH2-terminal tryptophan in adrenocorticotrophic hormone and melanocyte-stimulating hormone granules of adenohypophysis.

Fluorescence microscopy has demonstrated formaldehyde-ozone-induced fluorescence in the pars intermedia cells (melanocyte-stimulating hormone cells) and in certain cells of the pars distalis of the mammalian pituitary. From histochemical and chemical evidence the fluorescence is believed to reflect the presence of peptides with NH2-terminal tryptophan. In the pars distalis of hamster, cat and pig pituitary, the cells that exhibit formaldehyde-ozone-induced fluorescence have now been identified as adrenocorticotrophic hormone (ACTH) cells by immunohistochemistry. Granules from pig pituitaries were purified by passage through a succession of Millipore filters followed by centrifugation on a continuous sucrose gradient. Two granular fractions were identified by electron microscopy and found to contain high concentrations of peptides with NH2-terminal tryptophan as well as high ACTH bioactivity. These fractions, when pelleted and analyzed histochemically, displayed formaldehyde-ozone-induced fluorescence and ACTH-like immunoreactivity.

CYTOLOGICAL CHANGES IN THE MOUSE ANTERIOR PITUITARY AFTER NEONATAL THYMECTOMY: A LIGHT AND ELECTRON MICROSCOPICAL STUDY

SUMMARY A detailed light and electron microscope examination of the anterior pituitary gland of mice at different times after neonatal thymectomy was performed. Neonatal removal of the thymus resulted in a progressive increase in the number of somatotrophic hormone-producing cells showing characteristic changes represented by extremely distended cisternae of the endoplasmic reticulum and reduction in the number of hormone granules. These modifications were not observed in sham-operated controls or in neonatally splenectomized mice. The functional significance of these changes in relation to thymectomy and the significance of somatotrophic hormone for the development of the thymus and for immunological maturation is discussed. It is suggested that somatotrophic hormone and possibly other hormones determine certain specific steps of differentiation of precursor cells to immunocompetent cells and that the thymus is an endocrine target gland of somatotrophic hormone.

ULTRASTRUCTURE OF THE NEUROHYPOPHYSIS AS SHOWN BY FREEZE-ETCHING

SUMMARY Freeze-etched preparations of rabbit neurohypophysis and isolated hormone granules from cow neurohypophyses were examined electron microscopically. Many features seen in conventionally prepared sections were also apparent in freeze-etched replicas, particularly the neurosecretory nerve-endings containing elementary granules and elongated pituicyte processes. Measurements of diameters of both isolated and hormone-containing granules in situ indicated that they were slightly larger in freeze-etched material than in that prepared by conventional electron microscopic techniques. Fractures through the cores of hormone-containing granules showed a lamellar internal structure of 4 nm periodicity in both species examined.