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At least 199 records · Page 11Linked to original sources
PubMed1990

Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture.

The rat gene encoding phenylethanolamine N-methyltransferase (PNMT) was cloned and a consensus sequence for a glucocorticoid response element (GRE) was found at -513 bp, 5' to the transcriptional start site. In order to define the function of this element, fusion genes containing the PNMT promoter and a chloramphenicol acetyltransferase (CAT) reporter gene were constructed. These constructs did not express after transfection into any of 7 continuous cell lines, none of which endogenously produce PNMT. A system for transfecting chromaffin cells in primary culture was therefore devised using constructs containing 200 bp of the proenkephalin (ENK) promoter, whose expression characteristics are well known. pENK beta GAL-1, containing the ENK promoter with a lac Z reporter, was introduced into these cells and beta-galactosidase activity was visualized in situ. Approximately 90% of cells transfected were chromaffin; transfection efficiency was 5%. High levels of CAT activity were measured in chromaffin cells transfected with pENKAT12, possessing a CAT reporter. In contrast to tumor cell lines, pENKAT12 induction in these cells by forskolin and phorbol esters did not require a phosphodiesterase inhibitor. In this chromaffin system, both basal and regulated expression of the PNMT fusion genes were detected. Dexamethasone (dex) induced expression of pPNMT3000 and pPNMT900, containing the putative GRE and 3000 bp or 863 bp of PNMT promoter sequence, 4- to 10-fold. Expression of pPNMT300 and pPNMT100, which lack the GRE and contain 273 bp or 99 bp of PNMT promoter sequence, was unaffected by dex. Addition of the PNMT region spanning -490 to -863 bp conferred full dex responsiveness to a thymidine kinase promoter. Deletion of the putative GRE sequence by site-directed mutagenesis abolished the dex response. These data identify the sequence at -513 bp in the rat PNMT gene as a functional, positively acting GRE. Primary cultures of bovine chromaffin cells provide a biologically relevant expression system for transcriptional studies of catecholamine genes and their related neuropeptides.

Animals↗
PubMed1965

THE ADENOSINE-TRIPHOSPHATASE ACTIVITY OF ADRENAL CHROMAFFIN GRANULES.

1. The preparation of a fraction containing highly purified chromaffin granules from the bovine adrenal medulla is described. 2. The fraction contains an adenosine-triphosphatase activity that is stimulated by Mg(2+) and that cannot be explained by contamination with mitochondria or microsomes. 3. It is suggested that the adenosine-triphosphatase activity is related to the uptake of cate-cholamines by the chromaffin granules.

Adenosine↗
PubMed1985

The use of monoclonal antibodies in the study of the interaction between adrenal medullary cell membranes and chromaffin granules.

Simultaneous incubation of bovine adrenal medullary plasma membranes (PM) with chromaffin granules (CG) resulted in the release of the soluble granular content. The molecular mechanism of this process was studied with several monoclonal antibodies (mAb) raised against different plasma membrane components. Specific inhibition of the catecholamine secretion was obtained upon incubation with the monoclonal antibody UIA/NEU/VI B17. The corresponding antigen had an apparent molecular weight of 54000 Dalton. These results suggest a specific recognition between proteins located on the plasma membrane and chromaffin granule membrane, the interaction of which mediates exocytosis.

Adrenal Medulla↗
PubMed1990

Substance P enhances desensitization of the nicotinic response in bovine chromaffin cells but enhances secretion upon removal.

A fundamental process in neurosecretion is desensitization, or a declining response to a stimulus. The response of chromaffin cells to continuous nicotinic stimulation, secretion of catecholamines, desensitizes within a few minutes. The neuropeptide substance P (SP) has been reported to prevent desensitization in culture dish experiments and to enhance desensitization in patch clamp studies. In the present study, these contradictory responses have been demonstrated and the apparent contradictions resolved. We have measured catecholamine secretion by on-line electrochemical detection in a constant-pressure flow system. Isolated chromaffin cells cultured on quartz plates were stimulated with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in the presence and absence of SP. SP inhibited secretion and increase the rate of desensitization compared with stimulation by DMPP alone. However, when the cells were stimulated a second time with DMPP alone immediately after 5-min stimulation with SP + DMPP, the rate of desensitization was markedly lower than the control. Removal of SP after a desensitizing stimulation with SP + DMPP caused a slow secondary release of catecholamine in response to the continued stimulation with DMPP. The kinetic analysis of the secretory response shows that the primary response to SP is enhanced desensitization, but that upon removal of SP the response to DMPP desensitizes less rapidly. We suggest that SP protects some receptors from nicotinic desensitization while holding them in an inactive state, and that upon removal of SP these receptors can slowly respond to DMPP.

Animals↗
PubMed1990

Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell-free system.

In adrenal chromaffin cells, stimulation of Ca2+ influx leads to the secretion of neurotransmitters. The intracellular Ca2+ target involved in the fusion of secretory vesicles with the plasma membrane (PM) is still not known. We have reconstituted this fusion in vitro by using chromaffin granules (CGs) and target membrane vesicles and a Ca2(+)-dependent phospholipase A2 (PLA2). Vesicle fusion is measured by a fluorescence dequenching assay with octadecyl rhodamine B used as the marker. CGs fuse with PM vesicles only in the presence of active PLA2. The kinetics of this fusion process depend on the amount of target PM added. Once fusion competence of PM vesicles is achieved by exposure to PLA2 (primed PM vesicles), it is conserved after removal of the PLA2 even in Ca2(+)-free buffer. The kinetics of fusion between these primed PM vesicles and CGs depend on the amount of PM and on the temperature. Further incubation of the PLA2-treated PM vesicles at 30 degrees C in the absence of calcium results in an enhanced fusion competence. During this incubation, the amount of free arachidonic acid liberated by PLA2 decreases, suggesting that during a second process arachidonic acid may be processed to the terminal fusogen. The final steps of secretion can thus be subdivided into a Ca2(+)-dependent and -independent process: first, a Ca2(+)-dependent activation of PLA2 liberating fatty acids from phospholipids and second, a Ca2(+)-independent processing to the terminal fusogen and subsequent Ca2(+)-independent fusion of the CGs with the PM.

Adrenal Medulla↗