Constancy of DNA content in adrenal medulla nuclei of cold-treated rats.

Reports of changes in DNA content of certain types of cells following exposure to conditions of stress has led to the suggestion that two kinds of DNA may be present. One is genetic DNA, and the other is called "metabolic" DNA. In a further attempt to investigate the possibility of this phenomenon, determinations of DNA content were made on Feulgen-stained nuclei of adrenal glands and kidneys in cold-treated rats. Feulgen-stained nuclei were measured by two-wavelength microspectrophotometry. Particular attention was given to the handling of the smears in hydrolysis and staining. Mean values of Feulgen-DNA contents in a total of 720 nuclei demonstrated (a) a constancy of DNA content within 2% in individual nuclei both in adrenal medulla and kidney cortex, (b) no more than an average of 2% difference in DNA content between control and experimental nuclei, and (c) no more than an average of 1.5% difference in DNA content between normal kidney cortex nuclei and normal adrenal medulla nuclei. These results confirm the view that the more precise the measurement, the more accurately the constancy rule is obeyed. Moreover, there is no support for the concept of a metabolic DNA in the rat adrenal medulla.

Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway.

We report on the biochemical and immunological properties as well as on the cellular and subcellular distribution of two proteins, called secretogranins I and II. These proteins specifically occur in a wide variety of endocrine and neuronal cells that package and sort regulatory peptides into secretory granules. Both secretogranins take the same intracellular route as the peptides and are also sorted into secretory granules. Secretogranins I and II are biochemically and immunologically distinct proteins and differ from chromogranin A. Yet, these three proteins are similar to each other in many respects and therefore constitute one class of proteins. A remarkable feature of this protein class is a very acidic pI, brought about by a high content of acidic amino acids as well as by phosphorylation on serine and sulfation on tyrosine and O-linked carbohydrate. As a result, this class of proteins has a high net negative charge even at the acidic pH of the trans Golgi cisternae. We discuss the possibility that this property of the proteins may point to a role in the packaging of regulatory peptides into secretory granules.

Localization and characterization of carbohydrates in adrenal medullary cells.

The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.

An about 50,000-dalton protein in adrenal medulla: a common precursor of [Met]- and [Leu]enkephalin.

A protein that may be an enkephalin precursor has been identified in extracts of bovine adrenal medulla. This protein (about 50,000 daltons) appears to contain seven copies of [Met]enkephalin and one copy of [Leu]enkephalin. Digestion with trypsin and carboxypeptidase B yields [Met]enkephalin and [Leu]enkephalin in a ratio of almost 7 to 1. The enkephalins were identified by chromatography and by their binding to opiate receptors. Some characteristics of several other adrenal peptides that may serve as intermediates in the biosynthesis of the enkephalins are presented.

Identification of pro-opiomelanocortin-derived peptides in the human adrenal medulla.

Extracts from adult human adrenals contained high concentrations of immunoreactive beta-endorphin and alpha-melanotropin. Lower quantities of immunoreactive adrenocorticotropic hormone could also be detected. Distribution studies showed the presence of pro-opiomelanocortin fragments in the adrenal medulla. No alpha-melanotropin, beta-endorphin, or adrenocorticotropic hormone could be found in adrenal extracts from several other mammalian species. Analysis of the beta-endorphin-like immunoreactivity using region specific radioimmunoassays interfacing with gel filtration and reverse-phase high-performance liquid chromatography showed the majority of the beta-endorphin-like material to exist as nonacetylated beta-endorphin-(1-31) with a small percentage of lipotropin-sized molecules. The alpha-melanotropin-like immunoreactivity cochromatographed on gel filtration and reverse-phase high-performance liquid chromatography with desacetyl alpha-melanotropin. The data suggest that pro-opiomelanocortin is expressed in the adrenal medulla of humans but is not detectable in the adrenal glands of many other mammalian species.

Involvement of adenosine 3',5'-monophosphate in the activation of tyrosine hydroxylase elicited by drugs.

Immediately after the injection of reserpine (16 micromoles per kilogram, intraperitoneally), aminophylline (200 micromoles per kilogram, intraperitoneally), and carbamylcholine (8.2 micromoles per kilogram, intraperitoneally), the concentration of adenosine 3',5'-monophosphate in adrenal medulla of rats is increased severalfold. The three drugs also cause a delayed increase of medullary tyrosine hydroxylase activity. Our results are consistent with the view that an increase of medullary adenosine 3',5'-monophosphate concentration is involved in the drug-induced increase of tyrosine hydroxylase activity in adrenal medulla. Experiments with tyramine (130 micromoles per kilogram, intraperitoneally) suggest that the increase of tyrosine hydroxylase activity and of adenosine 3',5'-monophosphate concentrations is independent of an increase in adrenal catecholamine turnover rate.

Methionine-enkephalin and leucine-enkephalin in human sympathoadrenal system and pheochromocytoma.

To elucidate the physiological and pathophysiological significance of methionine- and leucine-enkephalin (Met-and Leu-enkephalin, respectively) in human sympathoadrenal system, the contents of these peptides in normal human sympathetic nervous system, adrenal medulla, and pheochromocytomas were determined by specific radioimmunoassays combined with reverse-phase high-performance liquid chromatography. Met-enkephalin-LI and Leu-enkephalin-LI, respectively) were detected by radioimmunoassay in adrenal glands, adrenal medulla, stellate ganglia, sympathetic trunks, and celiac ganglia, and their contents in adrenal medulla were highest. Existence of authentic Met- and Leu-enkephalin was confirmed by reverse-phase high-performance liquid chromatography. Met-enkephalin was approximately 74% of Met-enkephalin-LI, whereas Leu-enkephalin was approximately 30% of Leu-enkephalin-LI in human adrenal medulla. The ratio of Met- to Leu-enkephalin was 2.6 in human adrenal medulla, whereas it was higher in sympathetic ganglia or trunks. In four cases of pheochromocytoma marked difference in Met- and Leu-enkephalin contents was found between medullary and extramedullary tumors. The contents were about three orders higher and the Met- to Leu-enkephalin ratio was lower in medullary than in extramedullary pheochromocytomas, reflecting the tissues where the tumors arose. These results suggest the physiological roles of Met- and Leu-enkephalin in sympathetic nervous system and adrenal glands and their pathophysiological significances in pheochromocytomas.

Apparent stronger expression in the human adrenal cortex than in the human adrenal medulla of Mr 170,000-180,000 P-glycoprotein.

A monoclonal antibody (MAb), MRK 16, specific to Adriamycin-resistant human myelogenous leukemia cell line K562, was used to examine whether the antigen molecules (P-glycoprotein) recognized by the MAb are present in the adrenals. The materials examined included 61 human adrenals and several cell lines. Immunohistochemical analysis revealed that almost all of the human adrenal specimens (59 out of 61) were stained positively with MAb MRK 16 and that the antigen was strongly expressed even in cases where anticancer agents had not been given. Immunoprecipitation showed that the Mr 170,000-180,000 glycoprotein was present in all of the adult adrenals but not in fetal and neonatal adrenals. Furthermore, fluorescence image analysis revealed that the P-glycoprotein was more strongly expressed in the cortex than in the medulla, showing a tendency to occur in cell clusters in the latter area. The cell lines derived from animal adrenals (SW-13, Y-1, and PC-12) showed no positive staining with MAb MRK 16. It is suggested that this glycoprotein may be related to maturation of the adrenal, in which it possibly plays a physiological role.

Expression and characteristics of a novel human osteosarcoma-associated cell surface antigen.

The characteristics of a new osteosarcoma-associated cell surface antigen were studied by means of two murine monoclonal antibodies, TP-1 and TP-3, which were found to bind to two different epitopes on the same antigen, a monomeric polypeptide with a molecular weight of approximately 80,000. Immunohistochemical studies showed that the antigen was present in all osteogenic sarcomas tested, in most cases of malignant fibrous histiocytoma, in two malignant hemangiopericytomas and in a few synovial sarcomas, but not in other main groups of sarcomas and nonsarcomatous malignancies. In normal tissues it was detected only in clusters of cells in the adrenal medulla and in proximal kidney tubules. Also endothelial cells in proliferating capillaries in placenta and in most tumors were stained. The antigen was absent in resting but present in actively proliferating osteoblastic cells. The epitopes were resistant to proteolytic and sugar-cleaving enzymes but sensitive to high temperatures and could not be detected in paraffin-embedded specimens. The tissue distribution and properties of the antigen show that it is different from the sarcoma-associated antigens previously studied. In contrast to previous findings with three other anti-sarcoma monoclonal antibodies, no correlation was found between serum alkaline phosphatase activity and the amount of TP-binding substances in the same sera. Nevertheless, an apparently complex association between alkaline phosphatase and the TP-binding antigen seems to exist. Thus, the Mr 80,000 antigen extracted from an osteosarcoma cell line showed enzyme activity, whereas TP-binding molecules precipitated from patient sera contained alkaline phosphatase activity only in a few of the cases studied. Altogether our data suggest that the antigen defined by the TP antibodies may be a marker of osteoblastic differentiation. The pattern of antigen expression in malignant tumors is unique, inasmuch as the antigen is found selectively in sarcomas and in all 31 osteosarcomas tested.

The chromaffin granule surface. Localization of carbohydrate on the cytoplasmic surface of an intracellular organelle.

Intact chromaffin granules from bovine adrenal medulla are shown to have complex carbohydrates on their external (cytoplasmic) surface. This is demonstrated by the facts (1) that granules can be agglutinated by wheat germ agglutinin, and (2) that significant amounts of sialic acid can be removed from the granule surface with neuraminidase. Glycoproteins located in the granule membrane, and not glycolipids, are the molecules that mediate wheat germ agglutinin agglutination. The possible involvement of granule surface carbohydrate in the process of exocytosis is discussed.

Gangliosides and neutral glycolipids of human adrenal medulla.

Glycolipids were isolated from human adrenal medulla by DEAE-Sephadex A-25 and Iatrobeads column chromatography. The lipid-bound sialic acid was about 234 microgram/g fresh tissue. The glanglioside fraction contained two major gangliosides which accounted for 93% of the total lipid-bound sialic acid. They were identified as GM3, N-acetylneuraminylgalactosylglucosylceramide and GD3, N-acetylneuraminyl N-acetylneuraminylgalactosylglucosylceramide on the basis of cochromatography with authentic standards, sugar composition analysis, and neuraminidase digestion. GM3, N-acetylneuraminylgalactosylglucosylceramide and GD3, N-acetylneuraminyl N-acetylneuraminylgalactosylglucosylceramide occurred in a ratio of approximately 3 : 2, and the ratio seemed to be rather constant irrelevant of age and sex differences. The neutral glycolipid fraction consisted of GL1a, glucosylceramide (18%), GL1b, galactosylceramide (23%), GL2a, lactosylceramide (27%), GL3, digalactosylglucosylceramide (20%), and GL4, globoside (12%). The major fatty acids of all these glycolipids were 16 : 0, 18 : 0, 22 : 0, 24 : 0 and 24 :1.

Analysis of the carbon-13 and proton NMR spectra of bovine chromaffin granules.

Natural abundance carbon-13 and proton NMR spectra of bovine chromaffin granules have been obtained and analyzed using computer simulation techniques. High resolution spectra show the presence of a fluid aqueous phase containing epinephrine, ATP and a random coil protein. The protein spectrum contains unusually intense resonances due to glutamic acid and proline and has been simulated satisfactorily using the known amino acid composition of chromogranin A. The lipid phase of chromaffin granules gives rise to intense, but very broad, resonances in the carbon-13 spectrum. Protons in the lipid phase are also observable as a very rapid component of the proton-free induction decay (T2 approximately equal to 15 microns). Linewidths of the carbon-13 spectra have been used to set upper limits on rotational correlation times and on the motional anisotropy in the aqueous phase. These limits show that the aqueous phase is a simple solution (not a gel) that is isotropic over regions much larger than solute dimensions. No gel transition is observed between -3 and 25 degrees C. The carbon-13 spectra are definitely inconsistent with a lipoprotein matrix model and chromaffin granules previously proposed by Helle and Serck-Hanssen ((1975) Mol. Cell, Biochem. 6, 127-146). Relative carbon-13 intensities of ATP and epinephrine are not consistent with the known 1 : 4 mol ratio of these components. This fact suggests that epinephrine and ATP are not directly complexed in intact chromaffin granules.

Purification and characterization of an inhibitor protein with cytochalasin-like activity from bovine adrenal medulla.

A protein preparation with cytochalasin-like activity has been obtained from bovine adrenal medulla. Analysis by electrophoresis in SDS-polyacrylamide gel and chromatography in a Sephacryl S-200 column indicated that the inhibitor activity coincided with a 90 000 dalton polypeptide. The inhibitor decreased high-affinity binding of [3H]cytochalasin B to actin nuclei, apparently by competing with the drug for the same binding site. At substoichometric levels, the inhibitor had a potent effect on actin filament elongation and on actin-dependent gelation of cell extracts in vitro. These results suggest that the inhibitor may be involved in the control of actin filament assembly and interaction in the adrenal medulla.

Catecholamine content of chromaffin granule "ghosts' isolated from bovine adrenal glands.

Studies on the mechanism of catecholamine transport into chromaffin granules is complicated by the release of endogenous catecholamines. To overcome this problem chromaffin granule ghosts have been prepared by many investigators by osmotic lysis of the granules which results in a loss of over 90% of the endogenous catecholamine. However, in the studies reported here, the resulting ghosts still contained 36 +/- 3.9 nmol epinephrine/mg of protein if they were lysed by passage through a Sephadex G-50 column preequilibrated with hypoosmotic media. This residual catecholamine was found to slowly diffuse out of the ghosts in a temperature-dependent process at a rate sufficient to interfere with kinetic analysis of catecholamine transport. Attempts to remove the endogenous catecholamine from the ghosts indicated that most of it could not be removed by further osmotic shock of freeze-thaw treatments, but that over 85% of it was released from the granules by incubating them at 30 degree C for 90 min or by dialysis with a 35 and 86% loss of rate of catecholamine transport into the ghosts, respectively. If the endogenous catecholamine was removed from chromaffin granule ghosts by preincubating them for 90 min at 30 degree C, the resulting ghosts transported catecholamine with a linear Lineweaver-Burk plot indicating a Km of 12 +/- 2 microM. In addition, the resulting ghosts did not leak catecholamines over a 10 min period at 30 degree C, and the transport of catecholamines was blocked by reserpine and enhanced with increasing pH from 6.0 to 8.5.

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study.

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.

Bovine adrenal medullary chromogranin A: studies on the structure and further evidence for identity with dopamine-beta-hydroxylase subunit.

1. Chromogranin A was purified by the use of polyacrylamide gel electrophoresis. The amino acid composition of chromogranin A appeared to be nearly identical to that reported by other investigators and, moreover, was confirmed to be similar to that of dopamine beta-hydroxylase. 2. Dansyl-end group analysis revealed the presence of leucine as the only amino-terminal residue and quantitative estimations showed the presence of two leucine residues per molecule of 77 000 molecular weight. 3. Tryptic and CNBr patterns were obtained. Data are in good agreement with the concept of two nearly identical polypeptide chains per chromogranin A molecule of mol. wt 77 000. Patterns were compared with those obtained in parallel dopamine beta-hydroxylase and support the idea that chromogranin A and the dopamine beta-hydroxylase subunit are identical. Digestion with leucine amino peptidase gave further additional evidence for this suggestion. 4. Chromogranin A appeared to be free of carbohydrates. No cross-reaction was detected between chromogranin A and rabbit antibody against bovine adrenal dopamine beta-hydroxylase.

Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla.

The effect of dexamethasone on enkephalin-containing (EC) peptide levels and preproenkephalin mRNA levels was determined in adrenal medullary explants (glands) from sham and hypophysectomized (hypox) rats. Culture for 4 days in serum-free medium without dexamethasone resulted in a 13- and 4-fold increase in EC peptide levels in sham and hypox glands, respectively. The addition of dexamethasone (10(-5) M) produced a 20- to 26-fold increase in EC peptides in sham and hypox glands. In serum free medium, hypox glands showed a concentration dependent increase in EC peptides with the ED50 for dexamethasone equal to 5.7 x 10(-7) M. Since the glucocorticoid antagonist RU486 partially blocked the rise in EC peptides in sham glands, it appears that the increase in EC peptides in sham glands in the absence of dexamethasone is a result of a higher concentration of endogenous corticosterone in sham compared to hypox glands. Dexamethasone resulted in a 6-fold increase in preproenkephalin mRNA in hypox glands cultured for 2 days. This increase was approximately proportional to the increase in EC peptides seen at 4 days. In serum free medium progesterone, testosterone, and deoxycorticosterone failed to increase EC peptides in hypox glands. These results indicate that glucocorticoid treatment is required for maximal proenkephalin gene expression and EC peptide biosynthesis in cultured glands.

Purification and primary structure of pro-aldosterone secretion inhibitory factor from bovine adrenal chromaffin cells.

This report documents the purification and the complete primary structure of bovine aldosterone secretion inhibitory factor precursor (pro-ASIF). ASIF-(1-103) contains at position 69-103 of its carboxy-terminal end the formely identified 35-amino acid biologically active form, hence confirming the endogenous character of ASIF in the adrenal medulla. Compared to atrial natriuretic factor (ANF)-related peptide precursors, bovine ASIF displays 65% homology at the carboxy-terminal while the remaining amino-terminal part shows much more variability. Bovine pro-ASIF exhibits 73% homology with porcine pro-brain natriuretic peptide (BNP), a situation reminiscent of the relationship of pro-ANF in various species. When ANF- and BNP-related COOH-termini of bovine, porcine, human, rat, and chicken are compared, it appears that bovine ASIF and porcine BNP are closely related and belong to the same family which however appears to be much more heterogenous than the ANF-related family. These results strongly suggest that bovine ASIF is encoded by a precursor gene similar to the gene of BNP but different from the one encoding ANF.

Purification of a sulfated secretory protein from the adenohypophysis. Immunochemical evidence that similar macromolecules are present in other glands.

A sulfated secretory protein (apparent molecular weight approximately 70 000; isoelectric point approximately 4.8) recently identified as a minor component of mammotroph granules [21] has been purified, by ion-exchange chromatography on DEAE-Sephadex followed by preparative slab gel electrophoresis, from homogenates of bovine anterior pituitary glands pulse-labeled with [35S] sulfate. The homogeneity of the final product was established by one-dimensional as well as by two-dimensional gel electrophoresis followed by fluorography to reveal labeled polypeptides. Specific antibodies were generated against the purified protein. Immunodiffusion and radioimmuno-labeling of polyacrylamide gels revealed that proteins immunologically related to the adenohypophyseal sulfated component, exist in other glands, such as the neurointermediate pituitary and adrenal medulla. We envisage the possibility that the adenohypophyseal sulfated component belongs to a family of proteins which might play a widespread, though not general role in the secretory process.

Selective localization of the parathyroid secretory protein-I/adrenal medulla chromogranin A protein family in a wide variety of endocrine cells of the rat.

Secretory protein-I (SP-I) of parathyroid glands and chromogranin A ( CGA ) of adrenal medullary chromaffin cells are chemically similar if not identical proteins. Both proteins are contained within secretory granules and appear to be cosecreted with granule contents, for example, in the parathyroid with PTH and in the adrenal with epinephrine and dopamine beta-hydroxylase. Antisera to bovine SP-I and porcine CGA , together with antisera to a variety of peptide hormones, were used in an immunofluorescence study of rat tissues in order to determine the probable distribution and cellular localization of these proteins. In addition to their previously demonstrated presence in parathyroid and adrenal cells, the SP-I/ CGA protein family was detected in cells of the thyroid that contained calcitonin and often SRIF but not thyroglobulin; in cells of the anterior pituitary staining for the alpha-subunit of TSH/FSH/LH but not in cells staining for GH, PRL, ACTH, or beta-endorphin; in pancreatic islet cells staining for SRIF and pancreatic polypeptide-related peptides, but not for insulin or glucagon; in the celiac and mesenteric ganglia in cells some of which contained SRIF; and in the gastric antrum in cells containing SRIF, but not gastrin. SP-I/ CGA was not detected in cells of the liver, kidney, parotid gland, or acinar pancreas or in the intermediate or posterior lobes of the pituitary. These results suggest that this protein family enjoys a widespread but highly restricted distribution in many different endocrine-peptide cells of the rat, many that are believed to be of the APUD cell series. The possibility is raised that SP-I/ CGA plays some physiological role in the secretory process or exerts an effect of its own in the periphery after secretion.