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Carbamylcholine-stimulated catecholamine release from adrenal chromaffin cells was completely inhibited by pretreatment of the cells for 10 min with 1 μM calmidazolium. Catecholamine release due to 55 mM K+ and ionophore A23187 was also inhibited by calmidazolium but less effectively than release due to carbamylcholine. Inhibition of release appeared to be due to an effect of calmidazolium on a step distal to Ca2+ entry, since the carbamylcholine-stimulated rise in the concentration of intracellular free calcium, monitored using quin-2, was unaffected by calmidazolium. The possibility was considered that calmidazolium inhibited secretion through an effect on protein kinase C rather than calmodulin. However, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no demonstrable effect on catecholamine release, arguing against a significant role for protein kinase C in secretion from adrenal chromaffin cells. These results give further support to the notion that calmodulin plays a role in the secretory process in chromaffin cells.

We have examined bovine adrenal chromaffin cells in culture for the presence of the microtubule-associated protein 2. Chromaffin cells could be identified in culture on the basis of their staining with antibodies directed against secretory granule proteins. Using immunoperoxidase staining microtubule-associated protein 2 immunoreactivity was demonstrated to be present in chromaffin cells but not fibroblasts in culture. Microtubule-associated protein 2 immunoreactivity was present in the cell body, processes and varicosities of the chromaffin cells. Microtubule-associated protein 2 polypeptides were shown to be present in an adrenal medullary homogenate but not chromaffin granule membranes by immunoblotting. The results indicate that the neuronal cytoskeletal polypeptide microtubule-associated protein 2 is present in adrenal chromaffin cells. The presence of microtubule-associated protein 2 in both neurons and chromaffin cells may be related to their common embryonic origin.

The tyrosine kinase substrate p36 (calpactin heavy chain) is a calcium-dependent membrane- and cytoskeletal-binding protein. Using an affinity-purified antiserum raised against the p36 heavy chain from bovine adrenal medulla, we have examined the cellular distribution of p36 in developing and adult cerebellum. From immunoblotting, the level of p36 in cerebellum was found to decline during development. In dissociated cell cultures of P4 cerebellum, all cell types were labeled by anti-p36. In vibratome sections from cerebella of P10 rats, anti-p36 stained Purkinje cell bodies strongly and all other cell types less strongly, with the exception of cells in the external germinal layer, which were unstained. By 18 days postnatally p36 was present at higher levels in Bergmann glia and astrocytes of the white matter. In sections of adult cerebella, anti-p36 staining was restricted to Bergmann glial processes and to the processes of a subclass of astrocytes in the granular layer and the white matter. At no developmental stage was anti-p36 staining detectable in axons or dendrites in vibratome sections. These results suggest that p36 plays a role in some aspect of cellular differentiation common to all cerebellar cell types and may have additional functions in astrocytes of the adult cerebellum.

The presence of tropomyosins in adrenal chromaffin cells was demonstrated by immunoperoxidase staining of cultured chromaffin cells by an anti-tropomyosin antibody. In immunoblotting the antibody labelled polypeptides of Mr 39000, 36500 and 31000 in 100000 × g supernatants and heat-stable fractions of adrenal medulla. The adrenal medullary tropomyosins did not comigrate with smooth muscle tropomyosins. The 39-kDa and 31-kDa polypeptides comigrated with polypeptides labelled by anti-tropomyosin in brain supernatants. Chromaffin granule membranes possessed a 39-kDa polypeptide that was labelled by anti-tropomyosin.

Stimulation of adrenal chromaffin cells results in a rise in the concentration of intracellular free calcium which initiates catecholamine secretion by exocytosis. An understanding of the molecular basis of exocytosis will require knowledge of the sites of action of calcium. A role for calmodulin has been implicated in secretion from chromaffin cells, and isolated granule membranes bind both calmodulin and a series of cytosolic proteins in a calcium-dependent fashion. Here, we demonstrate that one of the cytosolic granule-binding proteins with a relative molecular mass (Mr) of 70,000 (70K) is a form of the calmodulin-regulated actin-binding protein caldesmon, first isolated from smooth muscle. Cytoplasmic gels assembled from an adrenal medullary extract in the absence of Ca2+ contained actin and the 70K protein. The association of both of these proteins with the cytoplasmic gel was inhibited by a micromolar concentration of Ca2+. In addition, we have demonstrated that the 70K protein is localized at the periphery of chromaffin cells. These results are consistent with the notion that 70K protein (caldesmon) has a role in regulating the organization of actin filaments of the cell periphery during the secretory process.