Abstract.Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca2+-dependent actin filament severing proteins, in the cortical surface of the chromatfin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K+-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-~-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.

Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) have been identified in bovine adrenal medullary tissue using an HPLC method. The values obtained were 0.1 +/- 0.05 mumol/g of tissue for both compounds. The subcellular fraction where Ap4A and Ap5A were present in the highest concentration was chromaffin granules: 32 nmol/mg of protein for both compounds (approximately 6 mM intragranularly). This value was 30 times higher than in the cytosolic fraction. Enzymatic degradation of Ap4A and Ap5A, isolated from chromaffin granules, with phosphodiesterase produces AMP as the final product. The Ap4A and Ap5A obtained from this tissue were potent inhibitors of adenosine kinase. Their Ki values relative to adenosine were 0.3 and 2 microM for Ap4A and Ap5A, respectively. The cytosolic fraction also contains enzymatic activities that degrade Ap4A as well as Ap5A. These activities were measured by an HPLC method; the observed Km values were 10.5 +/- 0.5 and 13 +/- 1 microM for Ap4A and Ap5A, respectively.

Scinderin, a novel Ca2+‐activated actin filament‐severing protein, has been purified to homogeneity from bovine adrenal medulla using a combination of several chromatographic procedures. The protein has an apparent mol. wt of 79,600 +/‐ 450 daltons, three isoforms (pIs 6.0, 6.1 and 6.2) and two Ca2+ binding sites (Kd 5.85 x 10(‐7) M, Bmax 0.81 mol Ca2+/mol protein and Kd 2.85 x 10(‐6) M, Bmax 1.87 mol Ca2+/mol protein). Scinderin interacts with F‐actin in the presence of Ca2+ and produces a decrease in the viscosity of actin gels as a result of F‐actin filament severing as demonstrated by electron microscopy. Scinderin is a structurally different protein from chromaffin cell gelsolin, another actin filament‐severing protein described. Scinderin and gelsolin have different mol. wts, isoelectric points, amino acid composition and yield different peptide maps after limited proteolytic digestion by either Staphylococcus V8 protease or chymotrypsin. Moreover, scinderin antibodies do not cross‐react with gelsolin and gelsolin antibodies fail to recognize scinderin. Immunofluorescence with anti‐scinderin demonstrated that this protein is mainly localized in the subplasmalemma region of the chromaffin cell. Immunoblotting tests with the same antibodies indicated that scinderin is also expressed in brain and anterior as well as posterior pituitary. Presence of scinderin and gelsolin, two Ca2+‐dependent actin filament‐severing proteins in the same tissue, suggests the possibility of synergistic functions by the two proteins in the control of cellular actin filament networks. Alternatively, the actin filament‐severing activity of the two proteins might be under the control of different transduction and modulating influences.

Earlier work by us as well as others has demonstrated that filamentous actin is mainly localized in the cortical surface of chromaffin cell. This F-actin network acts as a barrier to the chromaffin granules, impeding their contact with the plasma membrane. Chromaffin granules contain alpha-actinin, an anchorage protein that mediates F-actin association with these vesicles. Consequently, chromaffin granules crosslink and stabilize F-actin networks. Stimulation of chromaffin cell produces disassembly of F-actin and removal of the barrier. This interpretation is based on: (1) Cytochemical experiments with rhodamine-labeled phalloidin indicated that in resting chromaffin cells, the F-actin network is visualized as a strong cortical fluorescent ring; (2) Nicotinic receptor stimulation produced fragmentation of this fluorescent ring, leaving chromaffin cell cortical areas devoid of fluorescence; and (3) These changes are accompanied by a decrease in F-actin, a concomitant increase in G-actin, and a decrease in the F-actin associated with the chromaffin cell cytoskeleton (DNAse I assay). We also have demonstrated the presence in chromaffin cells of gelsolin and scinderin, two Ca(2+)-dependent actin filament-severing proteins, and suggested that chromaffin cell stimulation activates scinderin with the consequent disruption of F-actin networks. Scinderin, a protein recently isolated in our laboratory, is restricted to secretory cells and is present mainly in the cortical chromaffin cell cytoplasm. Scinderin, which is structurally different from gelsolin (different pIs, amino acid composition, peptide maps, and so on), decreases the viscosity of actin gels as a result of its F-actin-severing properties, as demonstrated by electron microscopy. Stimulation of chromaffin cells either by nicotine (10 microM) or high K+ (56 mM) produces a redistribution of subplasmalemmal scinderin and actin disassembly, which preceded exocytosis. The redistribution of scinderin and exocytosis is Ca(2+)-dependent and is not mediated by muscarinic receptors. Furthermore, our cytochemical experiments demonstrate that chromaffin cell stimulation produces a concomitant and similar redistribution of scinderin (fluorescein-labeled antibody) and F-actin (rhodamine phalloidin fluorescence), suggesting a functional interaction between these two proteins. Stimulation-induced redistribution of scinderin and F-actin disassembly would produce subplasmalemmal areas of decreased cytoplasmic viscosity and increased mobility for chromaffin granules. Exocytosis sites, evaluated by antidopamine-beta-hydroxylase (anti-D beta H) surface staining, are preferentially localized in plasma membrane areas devoid of F-actin.

Scinderin is a Ca(2+)-dependent actin filament severing protein present in chromaffin cells, platelets and a variety of secretory cells. It has been suggested that scinderin is involved in chromaffin cell F-actin dynamics and that this actin network controls the delivery of secretory vesicles to plasma membrane exocytotic sites. Moreover, scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated cells. Here we describe the molecular cloning, the nucleotide sequence and the expression of bovine chromaffin cell scinderin cDNA. The fusion protein obtained cross-reacts with native scinderin antibodies and binds phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PIP2) and actin in a Ca(+)-dependent manner. Antibodies raised against the fusion protein produced the same cellular staining patterns for scinderin as anti-native scinderin. Nucleotide and amino acid sequence analysis indicate that scinderin has six domains each containing three internal sequence motifs, two actin and two PIP2 binding sites and has 63 and 53% homology with gelsolin and villin. These data indicate that scinderin is a novel member of the family of Ca(2+)-dependent F-actin severing proteins which includes gelsolin and villin.