Proteins from chromaffin granules promote survival of dorsal root ganglionic neurons: comparison with neurotrophins.

Neurotrophins are established survival and differentiation factors for sensory dorsal root ganglionic (DRG) neurons. We have previously shown that proteins from the secretory granules of adrenal chromaffin cells have a capacity to promote the survival of cultured chick DRG neurons. Using DRG neurons from embryonic day (E) 8 chick embryos we show now that this material is (i) as effective as nerve growth factor (NGF), (ii) additive to NGF, neurotrophin-3, or -4, (iii) unlikely to be a neurotrophin, since the survival promoting effect can not be blocked by K252b, a specific inhibitor of the signal transduction pathways of neurotrophin high affinity receptors, (iv) partially blockable by antibodies to transforming growth factor-beta (TGF-beta) 1/2/3, and (v) more potent than any other out of 30 cytokines tested individually, including fibroblast growth factor (FGF)-5, epidermal growth factor (EGF), TGF-alpha, platelet-derived growth factor (PDGF)-AB, insulin-like growth factors (IGF)-I and -II, leukemia inhibitory factor (LIF), TGF-beta, glial cell line-derived neurotrophic factor (GDNF), stem cell factor, granulocyte-colony stimulating factor (G-CSF), oncostatin M, tumor necrosis factor (TNF)-alpha, and interleukins (IL)-1 through -12. We conclude that chromaffin cells, which are known to receive a sensory innervation, can provide (a) trophic factor(s), which, in addition to neurotrophins, may be relevant for the maintenance of DRG neurons.

Monoclonal antibodies reactive with a monoamine transporter preparation purified from bovine adrenal chromaffin granule membranes.

There have been no reports of monoclonal antibodies reactive with vesicular monoamine transporters from any source. Western blotting and ELISA data obtained using polyclonal serum from a mouse immunized with a highly purified bovine chromaffin granule monoamine transporter preparation yielded data consistent with the presence of antibodies to the transporter. Hybridomas produced by polyethylene glycol fusion of spleen cells from the mouse with X63/Ag8.653 myeloma cells were screened in an ELISA using partially purified transporter as coating agent. Of the 1142 wells containing colonies, 14 were positive in the initial screen. Hybridomas from wells testing positive were transferred to 24-well plates, grown up, and rescreened. Those still testing positive were subcloned, and the resulting positive wells containing single colonies were grown up and stocked. Of the 6 positive clones that tolerated freeze/thaw (0.5% of the wells tested), 1 was IgG1 kappa, 2 were IgG2a kappa, and 3 were IgG2b kappa isotypes. Ascites fluid was generated in pristane-primed BALB/c mice using hybridomas that had been cloned 2-3 times, and antibodies purified on immobilized Protein A. Immunoreactivity with a mixture of these antibodies, or with only one of them, coincided with dihydrotetrabenazine (TBZOH) binding activity in fractions eluted from all columns employed in the transporter purification. Antibody from at least one of the clones was capable of removing [3H]TBZOH binding activity from a partially purified preparation of transporter. Monoclonal antibodies exhibiting these properties have not been reported previously.

Galanin and neuropeptide Y in chromaffin granules from the guinea-pig.

We have examined the subcellular distribution of galanin-like immunoreactivity, neuropeptide Y-like immunoreactivity and the catecholamines noradrenaline and adrenaline in the adrenal medulla from guinea-pigs. By differential centrifugation of the adrenal medulla homogenate the neuropeptides as well as the catecholamines sedimented in a 10,000 g pellet. This pellet was resuspended and further examined in discontinuous and continuous density gradients. In the discontinuous gradient the catecholamines peaked in the heavy bottom fraction, assumed to contain chromaffin granules. Galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity were also enriched in this fraction. However, both neuropeptides showed high levels of sedimentable material also in a fraction of intermediate density. In the continuous density gradient, the sum of sedimentable and soluble catecholamines showed peak values in two fractions corresponding to 1.07 and 1.47 M sucrose, respectively. The NA peak in the denser fraction was more pronounced than the corresponding A peak. Galanin-like immunoreactivity showed only one peak, in the fraction corresponding to 1.07 M sucrose. The data suggest that galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity are partly stored with catecholamines in chromaffin granules. However, galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity was also found in fractions lighter than those containing the bulk of the catecholamines.

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens.

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.

Lysosomal H+-translocating ATPase has a similar subunit structure to chromaffin granule H+-ATPase complex.

Subunit structure of the lysosomal H+-ATPase was investigated using cold inactivation, immunological cross-reactivity with antibodies against individual subunits of the H+-ATPase from chromaffin granules and chemical modification with N,N'-dicyclohexyl[14C]carbodiimide. The lysosomal H+-ATPase was irreversibly inhibited when incubated at 0 degrees C in the presence of chloride or nitrate and MgATP. Inactivation in the cold resulted in the release of several polypeptides (72, 57, 41, 34 and 33 kDa) from the membrane, which had the same electrophoretic mobility as the corresponding subunits of chromaffin granule H+-ATPase. Cross-reactivity of antibodies revealed that the 72, 57 and 34 kDa polypeptides were immunologically identical to the corresponding subunits of chromaffin granule H+-ATPase. Dicyclohexylcarbodiimide, which inhibits proton translocation in the vacuolar ATPase, predominantly labeled two polypeptides of 18 and 15 kDa, which compose the membrane sector of the enzyme. These results suggest that the lysosomal H+-ATPase is a multimeric enzyme, whose subunit structure is similar to the chromaffin granule H+-ATPase. The subunit structure of other vacuolar H+-ATPases, revealed by cold inactivation and immunological cross-reactivity, is also presented.

Preparation and characterization of anti-human chromogranin A and chromogranin B (secretogranin I) monoclonal antibodies.

Chromogranin A, chromogranin B/secretogranin I and chromogranin C/secretogranin II are acidic sulphated and phosphorylated secretory proteins present in a large number of endocrine and neuronal tissues. It has been suggested that these proteins may be useful immunohistochemical markers for human tumours of endocrine origin and their measurement in plasma has been proposed as a diagnostic tool in patients with these tumours. In order to obtain anti-human chromogranins/secretogranins antibodies for clinical applications, we immunized mice with whole chromaffin granules isolated from human pheochromocytoma. The immune sera analysed by two-dimensional immunoblotting were found to recognize chromogranins/secretogranins and other unidentified proteins and to react in immunocytochemistry with pheochromocytoma as well as with a number of endocrine cells of different types. Hybridoma supernatants obtained from the splenocytes of a hyperimmune mouse, screened with an enzyme-linked immunosorbent assay, were analysed by both immunocytochemistry and two-dimensional immunoblotting. By using this experimental approach we were able to identify several monoclonal antibodies against human chromaffin granule components. In particular, we have characterized one anti-human chromogranin A and one anti-human chromogranin B/secretogranin I monoclonal antibody which showed a very specific pattern both in immunocytochemistry and in two-dimensional immunoblotting.

Monoclonal antibodies to chromaffin cells can distinguish proteins specific to or specifically excluded from chromaffin granules.

I have prepared a number of monoclonal antibodies to chromaffin cell membranes. One of these antibodies recognizes a number of antigenically related proteins that are present in all tissues examined. In the adrenal, these proteins are completely excluded from chromaffin granules but are present in other subcellular membrane fractions. This non-granule membrane-specific antibody has been designated NG3. A second antibody, CG7, binds to a single protein which segregates specifically into chromaffin granules. The protein recognized by CG7 is cytochrome b561, or chromomembrin B, one of the major protein components of chromaffin granule membranes. CG7 also labels a protein (the identical cytochrome b561) in bovine posterior pituitary neurosecretory vesicle membranes indicating that it functions in both peptidergic and catecholaminergic secretory granules. These two monoclonal antibodies provide useful probes of both granule and extra-granule membrane proteins for studies of membrane trafficking in chromaffin cells.

Enkephalins are associated with adrenergic granules in bovine adrenal medulla.

The subcellular localization of enkephalins was studied in the bovine adrenal medulla. In the adrenal medulla enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg6-Phe7 and Met-enkephalin-Arg6-Gly7-Leu8) are found free and in the form of cryptic peptides included in larger precursors. Total Met-enkephalin immunoreactivity, which includes free and cryptic peptides, was determined after a sequential enzymatic treatment with trypsin and carboxypeptidase B. Total Met-enkephalin immunoreactivity, dopamine beta-hydroxylase and catecholamines were found to have a parallel distribution in the various subcellular fractions. The bulk of the total Met-enkephalin immunoreactivity (42%) was recovered in the large granule fraction. The large granule fraction also contained 38% of the total dopamine beta-hydroxylase activity, and 42% of the total catecholamines. Enkephalins are thus concentrated in the chromaffin granules. Chromaffin granules were also separated according to the method of Terland & coworkers into two fractions: one containing the dense noradrenergic vesicles and the other containing lighter adrenergic vesicles. Total Met-enkephalin immunoreactivity was restricted to the fractions containing the lighter adrenergic vesicles. In these fractions the molar ratio of adrenaline to total Met-enkephalin immunoreactivity was 97. This study is in accord with immunocytochemical observations which have indicated that enkephalins are located in adrenergic and not in the noradrenergic cells in the bovine adrenal medulla.

Exposure of an antigen of chromaffin granules on cell surface during exocytosis.

The synthesis rate of the membrane proteins of the catecholamine-storing vesicles (chromaffin granules) of the adrenal medulla is lower than that of the secretory proteins of the contents. Based on these results we proposed that after exocytosis the membranes of chromaffin granules are retrieved and are re-used for several secretion cycles (see also ref. 4). This concept of re-use of granule membranes has been further strengthened by the finding that exogenous markers which are taken up by secretory cells during stimulation can be traced to the Golgi region and to immature secretory organelles. However, one basic question remains: are the membranes of secretory organelles specifically and completely removed from the plasma membrane and if so, how fast is this process? By using an antiserum against a membrane glycoprotein of chromaffin granules we have now obtained quantitative data which demonstrate that during exocytosis this antigen becomes exposed on the cell surface and disappears again to a large degree within 30 min.

Immunochemical evidence for exocytosis in isolated chromaffin cells after stimulation with depolarizing agents.

After chemical stimulation with depolarizing agents (Ba2+ or Ca2+/carbachol) isolated living chromaffin cells display a drastically increased binding capacity for anti-DBH, distributed spotwise on or near the outer cell membrane. This effect is inhibited by noradrenaline; it is not evoked by the non-exocytotically releasing agents tyramine and reserpine. the effect of apparent externalization of DBH is paralleled by the observation of a DBH-dependent binding of 125I-labelled protein A upon the same depolarizing stimuli. These observations are discussed as possible evidence for exocytotic activities.