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Notes: Abstract: A sensitive and selective radioimmunoassay for chromogranin A (Chrg A) has been developed to quantitate content, release, and biosynthesis of this secretory protein in neuroendocrine tissues. An antiserum raised against Chrg A from bovine adrenal medulla was found to detect predominantly only the Mr 70–75 kilodalton Chrg A in its native form, allowing the use of this antiserum as a quantitatively specific probe for Chrg A in cell-free extracts of the adrenal medulla and chromaffin cells. Chrg A comprises about 10% of the total protein of the chromaffin cell. It is released in parallel with Met-enkephalin and catechol-amines from the bovine chromaffin cell in primary culture in response to nicotine and nicotinic cholinergic agonists. From 14 to 22% of total Chrg A is released from the cell during a 15-min exposure to a maximally stimulatory dose of nicotine (10–100 μM). Chrg A release on nicotinic stimulation is blocked by D-600 and hexamethonium to the same extent as Met-enkephalin and catecholamine release. The parallel time course and percent release of Chrg A and Met-enkephalin indicate that these secretory polypeptides are contained in, and released from, functionally identical cellular compartments. Chrg A and Met-enkephalin pentapeptide sequences are present in the chromaffin cell at a ratio of about 2:1, although Chrg A is far more abundant on a mass basis. Chrg A and Met-enkephalin biosynthesis appear to be differentially regulated within the chromaffin cell, since chronic treatment of cells with nicotine and forskolin causes an elevation of Met-enkephalin pentapeptide without a concomitant elevation of intracellular levels of Chrg A.

Notes: Abstract: Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.

Notes: Abstract: Dynamin proteins have been implicated in many aspects of endocytosis, including clathrin-mediated endocytosis, internalization of caveolae, synaptic vesicle recycling, and, more recently, vesicular trafficking to and from the Golgi complex. To provide further insight into the function(s) of dynamin in neuroendocrine cells, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation, immunoreplica analysis, and confocal immunofluorescence. We found that dynamin, presumably the dynamin-2 isoform, is associated specifically with the membrane of purified secretory chromaffin granules. Oligomerization state analysis by sucrose density velocity gradients indicated that the granule-associated dynamin is in a monomeric form. Immunoprecipitation experiments coupled to double-labeling immunofluorescence cytochemistry revealed that the granular dynamin is associated with a syntaxin component that is not involved in the granule-bound SNARE complex. The possibility that dynamin participates in the coupling of the exocytotic and endocytotic reaction through the building of a granular membrane subset of proteins is discussed.

Notes: Abstract: Several lines of evidence suggest that phosphorylated products of phosphatidylinositol play critical functions in the regulation of membrane trafficking along the secretory pathway. To probe the possible involvement of phosphatidylinositol 3-kinase (PI 3-kinase) in regulated exocytosis, we have examined its subcellular distribution in cultured chromaffin cells by immunoreplica analysis and confocal immunofluorescence. We found that the PI 3-kinase heterodimer consisting of the regulatory and catalytic subunits was associated essentially with the subplasmalemmal cytoskeleton in both resting and nicotine-stimulated chromaffin cells. Attempts to immunoprecipitate PI 3-kinase with anti-phosphotyrosine antibodies failed, suggesting that the activity of PI 3-kinase was not modulated by tyrosine phosphorylation and/or physical interaction with SH2-containing proteins in stimulated chromaffin cells. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a potent inhibitor of PI 3-kinase, produced a dose-dependent inhibition of catecholamine secretion evoked by various secretagogues. Furthermore, cytochemical experiments with rhodamine-labeled phalloidin revealed that LY294002 blocked the disassembly of cortical actin in chromaffin cells stimulated by a depolarizing concentration of potassium. Our results suggest that PI 3-kinase may be one of the important regulatory exocytotic components involved in the signaling cascade controlling actin rearrangements required for catecholamine secretion.

Notes: Abstract Bovine adrenal chromaffin cells were maintained in culture in Dulbecco's modified Eagle's medium containing 20% foetal calf serum and 10 units per ml of Nerve Growth Factor. Under these conditions, chromaffin cells developed up to five neurites per cell. The neurites showed lateral branches and varicosities along their trunk which ended with thick growth cone-like structures. Cultures of chromaffin cells were stained by indirect immunofluorescence with antibodies against (a) chromogranin A to follow the distribution of chromaffin granules, the catecholamine-storing organelles, and (b) tubulin, to study the microtubular system during outgrowth of neurites. Chromogranin A antibodies showed a very intensely staining punctate pattern, not randomly distributed but localized in neurites. Chromaffin granules were found to migrate from the cell body to reach neurite endings where they were densely packed. Intense staining was also observed in varicosities; a linear arrangement of granules was evident along neurite trunks. Tubulin antibodies decorated a complex network, clearly visible at the cell periphery and also in the growth cone-like structures, in the palm region of the growth cone. Colchicine treatment effected retraction of neurites and disappearance of organized microtubule networks; chromaffin granules were found in the perinuclear region of the cell. Some tubulin (0.2% of total membrane proteins) was found in the purified chromaffin granule membrane preparation; however, this tubulin is probably associated with contaminating plasma membranes. By the criteria of morphology and staining with antitubulin antibodies, adult bovine chromaffin cells in culture display characteristics similar to those of sympathetic neurones. In addition, they showed an exaggerated transport of granules. Adult bovine chromaffin cells in culture offer an excellent model for studying the role of microtubules and the contractile apparatus in relation to cell morphological changes and neurosecretion.

Notes: In the present study we investigated the regulation of Chromogranin A (CGA) and Chromogranin B (CGB) biosynthesis in bovine chromaffin cells maintained in primary culture. Cellular proteins were labelled with [35S]methionine and the incorporated radioactivity was used as an index of the synthesis rate. The radioactivity incorporated into CGA was determined by immunoprecipitation, and that into CGB was quantified by a dot immunobinding assay using specific antibodies. Incubation of cells with carbamylcholine, nigh K+ or histamine, three potent stimulators of catecholamine secretion in chromaffin cells, increased the rate of CGA and CGB synthesis. On the other hand bradykinin, angiotensin II and prostaglandin E2, which cause little secretion, also produced an increase in both CGA and CGB synthesis. These results suggest that in chromaffin cells, the biosynthesis of chromogranins is not closely linked to the secretory activity. Inhibition of protein kinase C by sphingosine or by long-term treatment with phorbol esters, completely abolished the synthesis of CGA and CGB induced by carbamylcholine, bradykinin and prostaglandin E2 but decreased only partially the stimulating effect of histamine. Thus, protein kinase C may not be the sole effector involved in the secretagogue-induced modulation of Chromogranin synthesis. Forskolin, an activator of adenylate cyclase had no effect on CGA synthesis, but significantly enhanced the incorporation of radioactivity into CGB. The effect of forskolin was not modified by protein kinase C inhibitors and was additive to that induced by phorbol esters indicating that cyclic AMP did not stimulate CGB synthesis through a protein kinase C-dependent pathway. These observations suggest that the biosynthesis of CGA and CGB in chromaffin granules is independently regulated.

Notes: Secretogranin M (Sgll), also called chromogranin C, is an acidic tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurones. Although less abundant than chromogranin A (CGA) and chromogranin B (CGB), Sgll is found in adrenal medullary chromaffin granules. In the present study we investigated the regulation of Sgll biosynthesis in bovine chromaffin cells maintained in primary culture. Cellular proteins were labelled with [35S]methionine and the heat stable chromogranin enriched fraction was isolated. Following electrophoretic separation, the 86 kDa Sgll band was identified by sequence analysis using the Edman degradation procedure. The radioactivity incorporated in the 86 kDa Sgll band was used as an index of the Sgll synthesis rate. We found that stimulation of chromaffin cells with nicotine and histamine and to a smaller extent with angiotensin II and bradykinin significantly enhanced the rate of Sgll synthesis. In contrast direct depolarization with K+ had no effect on Sgll synthesis suggesting that the raise of cytosolic calcium evoked by high K+ may not be sufficient to induce modifications in Sgll synthesis. The possible second messenger pathways involved in the control of Sgll biosynthesis were investigated by using protein kinase C and adenylate cyclase activators. We observed that 12-O-tetradecanoylphorbol 13-acetate (TPA) and forskolin increased the basal rate of Sgll synthesis. Incubation with both TPA and forskolin was required to obtain an effect comparable to that produced by nicotine or histamine suggesting that these secretagogues recruit both protein kinase C- and cyclic AMP-dependent mechanisms to stimulate Sgll synthesis. Our results indicate that within chromaffin granules, the rates of CGA, CGB and Sgll synthesis are independently regulated and suggest a close relationship between the cell secretory activity and the biosynthesis of Sgll.The biological role of Sgll is unknown but it has been suggested that the protein may function as a precursor of potentially bioactive peptides. Here we identified a Sgll derived peptide corresponding to the C-terminal residues 582–586 in the soluble core of purified chromaffin granules. This peptide was released together with catecholamines upon stimulation of cultured chromaffin cells indicating that the peptide was present within the storage complex of chromaffin granules and was not the result of some artefactual proteolytic degradation of Sgll during the course of granule purification. We propose that this peptide is a specific product of the post-translational processing of Sgll. By analogy with peptides derived from CGA and CGB, it may possess some specific biological activity that remains to be identified.

Notes: The effect of guanosine triphosphate analogues on catecholamine secretion from permeabilized bovine chromaffin cells was examined. Guanosine 5′-[γ-thio]triphosphate was demonstrated to produce a dual effect on calcium-evoked secretion, enhancing the release through a mechanism involving protein kinase C and inhibiting secretion by a protein kinase C-independent pathway. We propose that two functionally distinct G-proteins control the stimulus - secretion coupling in chromaffin cells.

Notes: Regulated secretion requires both calcium and MgATP. Studies in diverse secretory systems indicate that ATP is required to prime the exocytotic apparatus whereas Ca2+ triggers the final ATP-independent fusion event. In this paper, we examine the possible role of trimeric G proteins in these two steps of exocytosis in chromaffin cells. We show that in the presence of low concentrations of Mg2+, mastoparan selectively stimulates G proteins associated with purified chromaffin granule membranes. Under similar conditions in permeabilized chromaffin cells, mastoparan inhibits ATP-dependent secretion but is unable to trigger ATP-independent release. This inhibitory effect of mastoparan on secretion was specifically reversed by anti-Gαo antibodies and a synthetic peptide corresponding to the carboxyl terminus of Gαo. In contrast, mastoparan required millimolar Mg2+ for the activation of plasma membrane-bound G proteins and stimulation of ATP-independent secretion in permeabilized chromaffin cells. The latter effect was completely inhibited by anti-Gαi3 antibodies and a synthetic peptide corresponding to the carboxyl terminus of Gαi3. By confocal immunofluorescence and immunoreplica analysis, we provide evidence that in chromaffin cells Go is preferentially associated with secretory granules, while Gi3 is essentially present on the plasma membrane. Our findings suggest that these two trimeric G proteins act in series in the exocytotic pathway in chromaffin cells: a secretory granule-associated Go protein controls the ATP-dependent priming reaction, whereas a plasma membrane-bound Gi3 protein is involved in the late calcium-dependent fusion step, which does not require ATP.