Adrenal Chromogranin Research Articles

Spontaneously hypertensive rat Y chromosome increases indexes of sympathetic nervous system activity.

Previous studies from our laboratory have demonstrated that the Y chromosome from the spontaneously hypertensive rat (SHR) is responsible for a significant portion of the elevated blood pressure and also produces an earlier pubertal rise in plasma testosterone. We performed the following studies to determine whether the SHR Y chromosome raises blood pressure by sympathetic nervous system responses as measured by adrenal chromogranin A and plasma and tissue catecholamines. Male SHR from the University of Akron colony were studied from 5 to 20 weeks of age. Blood pressure was measured by tail-cuff, tail artery cannulation, and aortic telemetry (Data Sciences); acute (air stress) and chronic (territorial colony) social stressors were compared; blood was collected for determination of plasma catecholamines; and adrenal glands were analyzed at 15 weeks for catecholamines. Rats with the SHR Y chromosome had higher blood pressure and plasma norepinephrine than those with the normotensive Wistar-Kyoto (WKY) Y chromosome. However, the SHR Y chromosome did not significantly change responsiveness to acute or chronic stressors. Phentolamine and clonidine prevented the stress responses. Adrenal chromogranin A levels were elevated 37% and 40% and adrenal norepinephrine content 29% and 100% at 4 and 10 weeks of age, respectively, in rats with an SHR Y chromosome compared with WKY. Chemical sympathectomy normalized blood pressure in all strains and significantly reduced norepinephrine (36% to 41%) in all strains except in WKY, which already had a normal blood pressure. In conclusion, the SHR Y chromosome appears to increase the chronic sympathetic nervous system. A potential mechanism could be a Y locus that influences chronic sympathetic nervous system activity, which may reinforce neurohumoral factors and structural components of the vessel wall, accelerating the development of hypertension.

Catecholamine secretory vesicles. Augmented chromogranins and amines in secondary hypertension.

Chromogranins A and B are major soluble proteins in chromaffin granules. Their adrenomedullary content is increased in the spontaneously (genetic) hypertensive rat. Is augmented catecholamine vesicular storage of the chromogranins a specific feature of genetic hypertension? To explore this question, we measured chromogranin A immunoreactivity, using a novel, synthetic peptide radioimmunoassay, in rat adrenal medullas 4-6 weeks after induction of the two-kidney, one clip Goldblatt model of renovascular hypertension and in unmanipulated control animals. We also measured messenger RNAs of chromogranins A and B and dopamine beta-hydroxylase by Northern blot. Immunoreactive adrenal chromogranin A was 3.3-fold higher (p < 0.01) in clipped rat adrenals. Adrenal catecholamine concentrations and phenylethanolamine-N-methyltransferase activity were also higher in clipped rats. Adrenal dopamine beta-hydroxylase activity (both membrane-bound and soluble forms) and corticosterone (glucocorticoid) concentration did not significantly differ between the groups. Adrenal medullary chromogranin A messenger RNA levels in clipped rats were 3.2-fold higher (p = 0.029) than those in the control group, and chromogranin B messenger RNA levels were 4.6-fold higher (p = 0.05). Dopamine beta-hydroxylase messenger RNA levels were 2.9-fold higher (p = 0.038). Thus, augmented synthesis and storage of adrenomedullary chromogranins A and B, catecholamines, and their biosynthetic enzymes appear to be characteristic of both acquired and genetic hypertension.

Fluorescence studies of nucleotide interactions with bovine adrenal chromogranin A

The binding of the fluorescent probe bis-ANS to chromogranin A, the major protein of adrenal chromaffin vesicles, caused a marked enhancement and blue shift in the fluorescence emission spectrum. The emission maximum shifted from 515 nm to 480 nm and the yield increased approx. 75-fold upon addition of 10 μM chromogranin A to 1 μM bis-ANS. Adenine nucleotides had clear effects on the bis-ANS fluorescence signal, while other nucleotides such as GTP, UTP and CTP had no discernible effect. Specifically, ATP caused a decrease in the fluorescence, whereas ADP and AMP caused a fluorescence increase. These results indicate adenine nucleotide binding to chromogranin A. Substitution of ATP with ε-ATP, an ATP derivative with a modification on the six-membered ring of the adenine base, failed to reduce the fluorescence intensity. Therefore, it was concluded that adenine bases play an important role in the chromogranin A-adenine nucleotide interaction.

Secretion of sulfated and nonsulfated forms of parathyroid chromogranin A (secretory protein-I).

Chromogranin A (secretory protein-I) is an acidic, sulfated glycoprotein found in secretory granules of most endocrine cells but not in exocrine or epithelial cells. Parathyroid chromogranin A is sulfated on tyrosine residues, whereas adrenal chromogranin A appears to be sulfated mainly on oligosaccharide residues. Chromogranin B, on the other hand, is tyrosine-sulfated in the bovine adrenal whereas this protein is absent from the parathyroid. The role of this tissue- or species-specific sulfation of chromogranin is not known. Tyrosine sulfation is a common post-translational modification of proteins in the exocytotic pathway and has been suggested to play a role in the sorting or intracellular transport of secretory proteins. To test this, porcine parathyroid tissue slices were metabolically labeled with 35SO4 and [3H]Lys, and the tissue and incubation medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoprecipitation with chromogranin A-specific antiserum or by radioimmunoassay for parathormone. Secretion of total and 3H-labeled chromogranin A was about 3- and 7-fold higher, respectively, at 0.5 mM than at 3.0 mM Ca2+, and secretion of 35SO4-labeled chromogranin A was 67-fold higher. This indicates that either sulfated chromogranin A is directed primarily to the Ca2+-regulated pathway or that sulfation occurs following sorting to this pathway. Sodium chlorate (1-10 mM) inhibited sulfation in a dose-dependent manner by up to 95% but it had no effect on the onset or rate of chromogranin A secretion. These data indicate that regulated secretion of parathyroid chromogranin A does not require sulfation of tyrosine residues.

Immunological studies on the occurrence and properties of chromogranin A and B and secretogranin II in endocrine tumors.

We investigated a variety of endocrine tumors for the presence of chromogranins A and B and secretogranin II. These antigens were identified by one- and two-dimensional immunoblotting and in some cases by immunohistochemistry. An antigen corresponding in electrophoretic behavior to adrenal chromogranin A was present in all types of tumors, including insulinomas, oat cell carcinomas, and Merkel cell tumors of the skin. Chromogranin B had a much more limited distribution. This antigen could not be detected in parathyroid adenomas, oat cell carcinomas, or Merkel cell tumors, either by immunoblotting and immunohistochemistry. The occurrence of secretogranin II was similar to that of chromogranin B, with the exception of a positive reaction in Merkel cell tumors. In benign pheochromocytomas, all three antigens were found consistently; whereas in two of three malignant pheochromocytomas, chromogranin B was absent. Our study establishes that in most cases chromogranins and secretogranin in tumors are identical to the adrenal antigens, but that these antigens are not always stored together. Chromogranin A is the most widely distributed marker for endocrine tumors.

The molecular cloning of the chromogranin A-like precursor of β-granin and pancreastatin from the endocrine pancreas

The cDNA encoding the precursor form of the chromogranin A-related proteins, β-granin and pancreastatin, was obtained by immune screening of rat insulinoma and pancreatic islet cDNA libraries. The sequence was virtually identical to that of rat adrenal chromogranin A, suggesting that the different molecular forms of chromogranin A immunoreactivity found in adrenal medulla and endocrine pancreas are related to differences in post-translational proteolytic processing. The rat chromogranin A, unlike its bovine and human counterparts, contained a 20-residue glutamine sequence inserted within the N-terminal β-granin sequence. Although the encoding CA(G/A) repeat recurs frequently in the rat genome, the rat chromogranin A molecule appears to be the product of a single gene and mRNA transcript.

Chromogranin A and B and secretogranin II in bronchial and intestinal carcinoids

Carcinoid tumours (bronchial and intestinal) were analyzed by immunoblotting for the presence of chromogranin A, B and secretogranin II. In all tumours an antigen corresponding in electrophoretic behaviour to adrenal chromogranin A was present. Lung carcinoids (3 out of 5) contained a relatively high concentration of a proteoglycan form of this antigen in addition. Chromogranin B was found in all tumours. In one and two dimensional immunoblotting it appeared identical to the corresponding adrenal antigen. Secretogranin II was also present, however concentrations (especially in intestinal carcinoids) were low and variable. Furthermore, in intestinal tumours it differed from the adrenal antigen by having a slightly higher molecular size and a more alkaline pI. Immunohistochemistry revealed that the tumour tissues stained positively for all three antigens. For secretogranin II the staining in intestinal tumours was relatively weak and quite variable. These results should provide a defined basis for immunohistochemical screening of carcinoids for the chromogranin/secretogranin antigens.

Structural characterization of adrenal chromogranin A and parathyroid secretory protein-I as homologs

We have isolated and purified adrenal chromogranin A (Ch A) for the purpose of making structural comparisons to parathyroid secretory protein-I (SP-I), because our earlier data indicated these two molecules may be the same protein. An improved purification step, using high-performance liquid chromatography (HPLC), has enabled us to demonstrate that both SP-I and Ch A consists of two species, one of approximately 72,000 Da and one of approximately 66,000 Da. The amino acid composition is the same for all four species. The difference in molecular mass is assumed to be due to carbohydrate content. Cyanogen bromide digestion of each of the four samples, followed by HPLC separation of the generated peptides, resulted in a chromatographic profile that was the same for each digest. Amino acid analysis of the eight peptide fragments obtained from each digest indicates that both species of Ch A and both species of SP-I yielded the same peptide mixtures following this cleavage reaction. One large (~ 50,000 Da) CNBr peptide was obtained and seven smaller ones, one of which contains cysteine. The large fragment behaved similarly to the intact molecule in a radioimmunoassay. HPLC separation of tryptic digests of Ch A (72,000 Da) and SP-I (72,000 Da) also resulted in elution profiles that were very similar to each other. Amino acid analysis revealed 23 peptides common to each digest. Ch A contained four peptides ranging in size from 4 to 30 residues that were not observed in the SP-I digest. SP-I contained two peptides, each with about 30 residues, that were not found in the Ch A digest. Nothing unusual was noted in any of the uncommon peptides. Thus, both a chemical and an enzymatic digestion of these molecules followed by analysis of the peptides generated, indicates that SP-I and Ch A are nearly identical homologs.