Chromaffin Cells In Primary Culture Research Articles

Gamma-aminobutyric acid uptake and localization in bovine chromaffin cells in primary culture

γ-Aminobutyric acid (GABA) uptake was studied in bovine chromaffin cells maintained in primary culture. Uptake was found to be dependent on Na + but not on K + and Ca 2+ ions; it was found that 2 Na + ions were necessary for each molecule of GABA transported. 2,4-Dinitrophenol, ouabain and vanadate inhibited GABA uptake showing the energy dependency of the system. Two affinity sites were demonstrated, a high affinity site and a low affinity site with K m values of 10 μM and 170 μM, respectively. While the low affinity site did not show large variations with culture age, the K m of thehigh affinity site increased from 1 μM in freshly isolated cells to 10 μM in 3–9 day-old cells. GABA uptake was unaffected by glutamic acid, aspartic acid, glycine and catecholamines, while taurine, betaalanine, nipecotic acid and l-2,4 diaminobutyric acid inhibited GABA uptake. Nipecotie acid and l-2,4 diaminobutyric acid acted as competitive inhibitors modifying K m values of the high affinity site. Subcellular studies performed on [ 3H]GABA-loaded chromaffin cells showed that GABA was not in secretory granules but was recovered in the 100,000 g soluble fraction. The GABA uptake process associated with chromaffin cells may be an important mechanism for regulating the modulation of catecholamine secretion. In addition, the presence of GABA in the cytosol indicates that this molecule may be an effector of chromaffin cell activity in addition to modulating catecholamine secretion.

Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture.

Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.

(Met)enkephalin and carboxypeptidase processing enzyme are co-released from chromaffin cells by cholinergic stimulation

A carboxypeptidase B-like enzyme is involved in processing of proenkephalin in adrenal medulla. Nicotine stimulated the co-release of this enzyme with (Met)enkephalin pentapeptide from bovine chromaffin cells in primary culture. The ratio of enzyme activity/immunoreactivity was determined for the released carboxypeptidase to provide an index of the level of enzyme activity per unit number of enzyme molecules. The ratio for the Co ++-stimulated carboxypeptidase secreted into the cell culture medium upon nicotinic stimulation was 10.1 ± 1.02 (pmol Met-enkephalin formed per ng carboxypeptidase immunoreactivity), while the Co ++-stimulated carboxypeptidase in the soluble and membrane components of purified chromaffin granules had lower ratios of 5.46 ± 0.70 and 1.07 ± 0.13, respectively. Hexamethonium, a nicotinic receptor antagonist, blocked the nicotine-induced release of the carboxypeptidase processing enzyme and (Met)enkephalin. These data suggest that a pool of carboxypeptidase enzyme molecules at a high state of activation are present in functionally mature granules whose contents are released by nicotinic receptor stimulation.

Alternative modes of enkephalin biosynthesis regulation by reserpine and cyclic AMP in cultured chromaffin cells.

Exposure of bovine chromaffin cells in primary culture to 5 microM reserpine or 25 microM forskolin results in an increase in enkephalin peptide levels within 24-48 hr; 25 microM forskolin (or cholera toxin at 50 micrograms/ml) causes a 1.5- to 2-fold increase in enkephalin peptide levels, which is maximal after 48 hr of exposure and is totally blocked by addition of cycloheximide (0.5 microgram/ml). Reserpine (5 microM) elicits a 1.5- to 2-fold increase in enkephalin peptide levels within 24 hr, which is only partially blocked by cycloheximide. Chromatographic analysis of cellular extracts shows that forskolin increases levels of both [Met]enkephalin pentapeptide and high molecular weight enkephalin-containing peptides, while reserpine causes an increase in [Met]enkephalin pentapeptide and a concomitant decrease in high molecular weight enkephalin-containing peptides, suggesting enhanced conversion of enkephalin precursor(s) to the mature polypeptide hormone. Measurement of preproenkephalin messenger RNA (mRNAenk) by RNA blot hybridization with a cDNA probe for mRNAenk reveals that forskolin and cholera toxin cause a relatively rapid (less than 17 hr) 3- to 5-fold increase in mRNAenk, while exposure to reserpine elicits a gradual decrease in enkephalin mRNA (a 50%-80% decline) beginning within 24 hr and continuing over a 72-hr period. These results suggest that forskolin and reserpine differentially regulate enkephalin biosynthesis in cultured chromaffin cells, the former by increasing, presumably via a cAMP-dependent mechanism, cellular mRNA coding for preproenkephalin and the latter by a post-translational increase in proenkephalin processing.