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At least 217 records · Page 12Linked to original sources
PubMed1991

Expression and processing of mouse proopiomelanocortin in bovine adrenal chromaffin cells. A model system to study tissue-specific prohormone processing.

Many neuroendocrine precursor proteins, such as proopiomelanocortin (POMC), are cleaved in a tissue specific manner at distinct pairs of basic amino acids. Elucidating the specificity of the prohormone endoprotease(s) is essential to understanding cleavage specificity. However, isolation of these enzymes has been difficult, due to the inability to distinguish authentic maturation enzyme from the many other trypsin-like activities present in tissue homogenates. Recently, a "signature" of the insulin cell endoprotease(s) was defined in vivo by assessing the processing of a series of mutant cleavage sites in a model prohormone, mouse POMC (mPOMC) (Thorne, B. A., and Thomas, G. (1990) J. Biol. Chem. 265, 8436-8443. To investigate mechanisms of tissue-specific processing, we sought to identify the endoprotease signature of a cell having a processing phenotype distinct from insulinoma cells. In this report, the cleavage site specificity of the endoprotease(s) expressed in bovine adrenal chromaffin cells is examined. High levels of mPOMC (1.6 pmol/10(6) cells) were expressed in these cells using a vaccinia virus vector, and the precursor was targeted to the regulated secretory pathway. Analysis of POMC-derived peptides revealed that chromaffin cells processed the prohormone to a set of peptides highly similar to anterior pituitary corticotrophs, including adrenocorticotropin hormone (ACTH) and beta-lipotropin, gamma-lipotropin, and beta-endorphin. This processing contrasted with the pattern of cleavage site utilization in Rin m5F insulinoma cells, which more closely resembled that of the intermediate pituitary melanotrophs. However, the processing preference for the sequences of pairs of basic amino acids (as tested using the entire series of mutant cleavage sites; -LysArg- (native), -ArgArg-, -ArgLys-, -LysLys-, -HisArg-, -MetArg- at the ACTH/beta-lipotropin junction and -LysLys- (native), -LysArg-, -ArgArg-, -ArgLys- in beta-endorphin) was the same in both insulinoma and adrenal chromaffin cells, suggesting recognition and cleavage by similar enzymes in both cell types. The cell-specific processing of mPOMC may thus result from expression of a common core set of processing enzymes and factors unique to each cell type affecting the enzyme accessibility to precursor cleavage sites.

Adrenal Medulla↗
PubMed1982

A model of biogenic amine accumulation into chromaffin granules and ghosts based on coupling to the electrochemical proton gradient.

A model is presented to explain catecholamine uptake into isolated chromaffin granules and ghosts. It is based on the following observations and conclusions: 1) the chromaffin granule maintains one of the lowest permeabilities to cations of any previously isolated subcellular organelle and the lowest permeability to protons, 2) a transmembrane proton gradient delta pH exists across the chromaffin granule membrane that is not media dependent, with a measured internal pH of 5.5; 3) the addition of ATP to a suspension of chromaffin granules results in the generation of a membrane potential of 80-100 mV, positive inside; 4) an H+-translocating ATPase responsible for the generation of this electrochemical proton gradient (delta micro H+) exists within the membrane of the chromaffin granule; 5) net amine accumulation proceeds in the presence of both a delta pH alone and a transmembrane potential gradient delta psi alone, with the maximal rate and extent occurring in the presence of a delta pH and a delta psi together (delta micro H+); 6) when catecholamine uptake is in equilibrium with the delta micro H+, the equilibrium distribution of amines (delta micro A) obeys the relationship delta micro A = delta psi-2Zdelta pH. The model, in its elemental form, states that an H+-translocating ATPase for the generation of a delta pH and delta psi exists within the membrane of the chromaffin granule. This electrochemical proton gradient provides the driving force for amine influx, which proceeds via a reserpine-sensitive antiport carrier mechanism coupled to proton efflux. The model is discussed in terms of its physiological implications and significance for other amine-containing subcellular organelles, with particular respect to the species of catecholamine transported, and other unresolved issues.

Animals↗
PubMed1990

[Melanotic paraneurons].

Histogenetic scheme of three germ layers causes difficulties in explanation of some tumorous structures derived from different classes. A conception of paraneurons concedes (besides "central" neuroectodermal paraneurons) a possibility of their local origin. The idea anticipates a broader phylogenetic ability of divergent differentiation of single extraneural cells. Paraneuronal theory used in oncology can explain local origin of some presumable combination variants of tumours, e.g. carcinoid type paraneuromas with melanin pigmentation. Substantial frequency of such tumours eliminates possibility of random coincidence and argues for ranging "peripheral melanin system" among paraneurons.

Chromaffin System↗
PubMed1989

Barium ions enter chromaffin cells via voltage-dependent calcium channels and induce secretion by a mechanism independent of calcium.

Barium ions enter chromaffin cells via voltage-sensitive calcium channels, although the intracellular site of barium action is distinct from that of calcium. The entry of barium primarily through voltage-sensitive channels was indicated by experiments showing inhibition of 133Ba2+ uptake by blockers of voltage-dependent calcium channels. In addition, 133Ba2+ uptake was stimulated by 50 mM KCl but not by nicotine. Furthermore, 133Ba2+ uptake was inhibited by hyperosmolarity, which specifically blocks the voltage-sensitive calcium channel but not the receptor-associated calcium channel. These conclusions from studies on barium uptake were also borne out by experiments measuring catecholamine secretion. Thus, blockers of voltage-dependent calcium channels which inhibited barium uptake also inhibited barium-induced catecholamine secretion. In other experiments, simultaneous stimulation with nicotine and barium in the presence of calcium evoked coincident and additive catecholamine secretion. By contrast, when 50 mM KCl was substituted for nicotine in the same experimental design, barium ions inhibited potassium-induced catecholamine secretion at low calcium concentrations. Only at high calcium concentrations were barium-induced and potassium-induced secretion additive. These data also indicate that barium and calcium compete at the voltage-sensitive pathway. Furthermore, these additivity data suggest that once inside the cell, barium and calcium have two distinct mechanisms of action. As predicted by this hypothesis, in digitonin-permeabilized chromaffin cells either calcium or barium stimulated catecholamine release, and in the presence of both cations catecholamine secretion was equivalent to the sum of secretion with either cation alone. Additional support of this concept comes from experiments showing that while calcium-mediated catecholamine secretion is sensitive to trifluoperazine and imipramine, barium-mediated secretion is not. Taken together, all these data indicate that there are two distinct intracellular sites of action for barium and calcium. In contrast to catecholamine secretion, non-exocytotic ascorbic acid secretion was induced by nicotine and potassium in the presence of calcium, but not by barium alone. These data provide additional evidence that barium acts by a different mechanism than calcium, in still another secretory system in chromaffin cells.

Animals↗
PubMed1979

Light scattering turbidity changes as a measure of the kinetics of Ca2+ -promoted aggregation of chromaffin granule membrane ghosts.

Changes in turbidity seen when chromaffin granule membrane ghosts are aggregated by Ca2+ can be modelled as dimerization of hollow spheres using Rayleigh-Gans-Debye light-scattering theory. The experimental changes agree well with the calculations. Thus, if shape or refractive index changes produced by osmotic perturbation, ion uptake, etc. can be excluded, turbidity readings can be used to follow the progress of the aggregation reaction of storage vesicles and other small particles or macromolecules.

Animals↗
PubMed1982

ATP-activated exchange of catecholamines by isolated intact chromaffin granules.

Isolated chromaffin granules were found to accumulate exogenous (R)-[3H]-norepinephrine at 37 C in the presence of Mg2+-ATP by a process involving one-to-one exchange for endogenous epinephrine. By contrast, omission of Mg2+-ATP from the incubation medium resulted in a substantial unidirectional efflux of the endogenous epinephrine independent of external catecholamine concentrations. However, the effects of ATP on the spontaneous efflux and the exchange process were found to occur with different kinetic parameters and differential sensitivity to a stilbene disulfonate inhibitor of the granule ATPase, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. These data lead us to conclude that ATP catalyzes bidirectional catecholamine flux across the granule membrane (not net accumulation) by an ATPase-dependent mechanism. In addition, ATP has the ability to suppress unidirectional efflux of catecholamines from granules by a mechanism independent of the ATPase or the exchange mechanism. The data also allow us to distinguish among three possible models for how these processes might be occurring.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1983

Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules.

Intact chromaffin granules isolated from bovine adrenal medulla were incubated at 37 degrees C for up to 22 hr. Processing of enkephalin-containing (EC) peptides in the granules was followed by the change in their size distribution as shown by chromatography on Sephadex G-75 columns. A gradual shift toward lower molecular weight EC peptides was observed during the incubation, indicating processing of the higher molecular weight to lower molecular weight EC peptides. The total amount of [Met]-enkephalin, free and in peptide linkage, remained constant indicating that little or no nonspecific degradation occurred during the experiment. HPLC resolution of the fraction containing the low molecular weight EC peptides showed that free enkephalins as well as [Met]enkephalin-Arg6-Phe7 and [Met]enkephalin-Arg6-Gly7-Leu8 accumulated while [Met]enkephalin-Arg6 and [Met]enkephalin-Lys6 disappeared. All the above data indicate the presence of an atypical trypsin activity and the presence of a carboxypeptidase B-like activity within the granules. From the rates of accumulation of the low molecular weight EC peptides and the disappearance of the higher molecular weight EC peptides, a processing rate of 65-70 pmol/g tissue per hr was estimated, which calculates to a lifetime of 6-8 days for EC peptides in the granules. Under steady-state conditions this rate of processing appears to be too low to produce significant amounts of free enkephalins from larger EC peptides. This is well in accord with previous observations that relatively small amounts of free enkephalins are found in bovine adrenal medulla.

Animals↗
PubMed1985

Organization and distribution of the rat subdiaphragmatic vagus and associated paraganglia.

This experiment analyzed the organization of the rat abdominal vagus. To spare delicate tissues and preserve positional information, untrimmed blocks of the subdiaphragmatic viscera (N = 22) were fixed, impregnated by using a pyridine-silver protocol, and double embedded. Each block was sectioned transversely at 7 micron, and a section every 70 micron from the diaphragm to the cardia was analyzed. The features of the section were traced and digitized for computer reconstruction. Included in the measurements were sizes and locations of bundles, fascicles, and paraganglia. The anterior and posterior vagi were consistently distinctive in size, distribution, cross-sectional shape, and paraganglionic content. In the most common pattern (41% of animals), the anterior trunk coursed longitudinally on the ventral surface of the esophagus, giving off at successively more distal levels the hepatic branch, the accessory coeliac branch and then the bundles of the anterior gastric branch. The posterior trunk separated into a coeliac branch and a posterior gastric branch, each consisting of numerous bundles, in the most distal quarter of the esophagus. Fifty-nine percent of all animals exhibited one or more significant variations in vagal organization (e.g., double primary trunks--41%, supernumerary branches--18%, or atypical branching sequences--9%). Four to 14 vagal paraganglia (mean = 8 +/- 1; equivalent to 32/rat, corrected for sampling) were found in each animal, and no branch was consistently devoid of paraganglia. Ninety-four percent of the paraganglia were located at nerve branch points. Some of the larger paraganglia contained at their central poles one to six neurons with soma diameters ranging from 14 to 22 micron.

Animals↗
PubMed1978

The endocrine nature of the paranganglia of man.

Brightly fluorescent paraganglia were found in the retroperitoneal tissue of adult man. The histofluorescence properties of the paraganglia indicate the presence of tryptophyl peptides, which might be of endocrine importance.

Animals↗