Calmodulin-Binding Proteins in Chromaffin Cell Plasma Membranes

Abstract: Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins ( 240, 105 , and 65 kilodaltons) were eluted by an EGTA-containing buffer. ¹²⁵I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common ( 65, 60, 53 , and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.     

Calmodulin-binding proteins in granule and plasma membranes from bovine chromaffin cells

Calmodulin-binding proteins in chromaffin granule membrane and chromaffin cell plasma membranes have been investigated and compared. Chromaffin granules were purified by centrifugation over a 1.7 M sucrose layer. Plasma membranes were obtained in a highly purified form by differential and isopycnic centrifugation. Enzymatic determinations of 5'-nucleotidase, a generally accepted plasma membrane marker, showed a 40-50-fold enrichment as compared to the cell homogenate. Marker enzyme studies demonstrated only minimal contamination by other subcellular organelles. After solubilization with Triton X-100, calmodulin-binding proteins were isolated from chromaffin granule membranes and plasma membranes by affinity chromatography on a calmodulin/Sepharose 4B column. On two-dimensional polyacrylamide gelelectrophoresis a prominent protein (Mr = 65,000, pI ranging from 5.1 to 6) consisting of multiple spots, was present in the calmodulin-binding fraction from chromaffin granule membranes as well as from plasma membranes. Besides this 65 kDa protein both fractions had at least four groups of proteins in common. Also, proteins typical for either preparation were observed. In the calmodulin-binding protein preparations from chromaffin granule membranes a prominent spot with Mr = 80,000 and a pH ranging from 5.0 to 5.7 was present. This protein was enzymatically and immunologically identified as dopamine-beta-monooxygenase.     

Membranes of chromaffin granules. Isolation and partial characterization of two proteins

Membranes of chromaffin granules were isolated from the adrenal glands of four different species. The solubilized membrane proteins could be resolved into several bands by polyacrylamide-gel electrophoresis (alkaline and acid gel systems). Two major protein components appeared to be common to the chromaffin granule membranes of ox, horse, pig and man. The various membrane proteins of bovine chromaffin granules were separated by filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. Two major membrane proteins (A and B) were obtained in purified form. Treatment of protein A with 2-mercaptoethanol before electrophoresis resulted in two more rapidly migrating subunits, whereas protein B was unaffected by mercaptoethanol treatment. The amino acid compositions of the two purified proteins were determined. They are very similar to that of the total membrane proteins but significantly different from that of the chromogranins, the soluble proteins of chromaffin granules.     

Similarities of physico-chimical and antigenic properties of neurocupreins and extremely acidic copper proteins from chromaffin granules

Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested that the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.     

Similarities of physico-chemical and antigenic properties of neurocupreins and extremely acidic copper proteins from chromaffin granules

Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested the the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.     

Isolation of a protein from the plasma membrane of adrenal medulla which binds to secretory vesicles.

Solubilized proteins of the plasma membrane of bovine adrenal medulla were fractionated on the basis of their affinity for secretory vesicles. The isolation procedure included preparation of a highly purified fraction of plasma membranes, its solubilization in detergent, and application to a column prepared from glutaraldehyde-fixed chromaffin granules. Using this technique, one major polypeptide (80% of the material bound) was isolated. This protein has been shown to originate from the plasma membrane and has no affinity for fixed bovine adrenal medullary mitochondria or lysosomes. It is eluted most effectively by low pH (3.0) and can be rebound and re-eluted from fixed secretory granules. In sodium dodecyl sulfate and beta-mercaptoethanol it has an apparent molecular weight of 51,000. In addition, two minor components, comprising about 20% of the material bound were detected having apparent molecular weights in sodium dodecyl sulfate of 14,000 and 62,000. It is suggested that such a molecule could function as a plasma membrane-located receptor for chromaffin granules during the secretory process.     

Biochemical studies of the chromaffin granule. III. Redistribution of lipid phosphate,dopamine-β-hydroxylase and chromogranin a after freezing and thawing of the isolated granule membranes

Freezing and thawing a dialysed suspension of lysed chromaffin granules and sedimented membrane preparations resulted in redistribution of lipid phosphate and protein. By this treatment the high ratios of lipid phosphate/protein in the membrane fragments, isolated on sucrose density gradient from the dialysed suspensions and the sedimented membrane preparation, were reduced from 1.56 to 1.03 μmoles/mg and from 1.97 to 0.83 μmoles/mg, respectively.Multilamellar, liposomal structures could be isolated from the frozen and thawed membrane preparations and were found to sediment in the 0.4 M sucrose layer by density gradient centrigugation. This fraction was without morphological resemblance to the intact chromaffin granules or their membranes and was found to account for 53% of the lipid phosphate, 35% of chromogranin A, 21% of dopamine-β-hydroxylase activity and 12% of the protein of the total preparation. The specific activities of chromogranin A and dopamine-β-hydroxylase in the artificially formed liposomal structures closely resembled that of the solubilized protein and was significantly higher than in the lipid phosphate-depleted membrane fragments recovered in the 1.1 M sucrose layers.It is concluded that freezing and thawing as a means of purifying the isolated granule membranes lead not only to the solubilization of chromogranin A, but also to removal of dopamine-β-hydroxylase activity and lipid phosphate from the labile membrane fragments.     

A Similar Calmodulin-Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.     

Acidic copper-containing proteins--an intermediate link to the electron transfer stage from cytochrome b-561 to dopamine-beta-monooxygenase

The interaction of acidic copper-containing protein from the membranes of chromaffin granules has been investigated with cytochrome b-561 and dopamine-beta-monooxygenase from the same source. By the use of spectral and polarographic measurements it was demonstrated that the acidic copper-containing protein acts as an electron acceptor for cytochrome b-561 and as electron donor in the reactions, catalyzed by dopamine-beta-monooxygenase. According to the data obtained the possible function of the acidic copper-containing protein in vivo on the area of electron transfer chain between cytochrome b-561 and dopamine-beta-monooxygenase are discussed. The activation or inhibition of the electron transfer reactions by a variety of phospholipids, analogs of membrane lipids of chromaffin granules has been established. The experiments were performed in a model systems by the use of highly purified preparations of proteins and bilamellar liposomes and micelles, prepared from the corresponding phospholipids.     

The distribution of Concanavalin A receptor sites on the membrane of chromaffin granules.

The distribution of concanavalin A (con A) receptor sites on the membranes of chromaffin granules has been investigated by binding studies using 125I-labelled con A and by electron-microscope studies using ferritin-labelled con A. In both experiments con A was observed to bind to chromaffin granule membranes but not to intact granules. The ferritin-con A particles bind to only one of the two possible surfaces of the chromaffin granule membranes. These results are in agreement with previous observations concerning the asymmetric distribution of saccharide residues on the surfaces of a number of different plasma membranes. They suggest that for the intracellular membrane of the chromaffin granule the saccharide sites, like those in plasma membranes, are not exposed to the cell cytoplasm. Further work is necessary to establish whether these sites are on the inner surface of the membrane or whether they are unmasked during the conversion of granules to membrane ghosts.     

A role for sialic acid containing substrates in the exocytosis-like in vitro interaction between adrenal medullary plasma membranes and chromaffin granules

The “in vitro” interaction between bovine adrenal medullary plasma membranes and chromaffin granules has recently been proposed as a putative cell-free model for exocytosis because calcium ions specifically control the plasma membrane-induced release of 10^?7 and 10^?5 M. Addition of ruthenium red or pretreatment with neuraminidase gradually blocks this interaction indicating that sialic acid containing substrates may be of major importance. These observations and similar results obtained by other authors working on different systems suggest a role for sialic acid containing moieties in exocytosis.     

Chromogranin C: A Third Component of the Acidic Proteins in Chromaffin Granules

We characterized a group of acidic proteins of bovine chromaffin granules with an antiserum raised against a protein described by Rosa and Zanini [Eur. J. Cell Biol. 31, 94-98 (1983)] in pituitary gland. In adrenal medulla the proteins reacting with this antiserum are confined to chromaffin granules. Their largest component has a Mr of 86,000 and a pI of 5.0. In addition six proteins of lower molecular weight are recognized by this antiserum. In a cell-free system only one protein is synthesized that can be precipitated with this antiserum. The properties of these proteins are very similar to those of the previously described chromogranins A and B; however, there is no immunological cross-reaction between these protein groups. We suggest this third group of acidic proteins of chromaffin granules be named chromogranins C.     

Isolation and Immunological Characterization of a Glycoprotein from Adrenal Chromaffin Granules

A glycoprotein (s-GP III) was isolated from the soluble lysate of chromaffin granules by chromatography with immunoaffinity and lectin columns. An identical protein (m-GP III) was shown to be present in the granule membranes. The apparent molecular weight of these glycoproteins as determined by the electrophoresis system of Laemmli (1970) was 43,000 under reducing conditions. In the absence of mercaptoethanol they aggregated to dimers. Antisera were raised against both the soluble and the membrane-bound forms of this glycoprotein. With these antisera GP III was further characterized: Immunoreplicas were obtained after two-dimensional electrophoresis of soluble and membrane-bound proteins of chromaffin granules. GP III was identified as a protein with a rather broad pI (4.6-5.3), indicating microheterogeneity. As shown by subcellular fractionation, m-GP III is specifically confined to chromaffin granules. GP III can therefore be used as a marker for the membranes of these organelles. The soluble form is secreted from adrenal medulla during stimulation with carbamylcholine chloride. An immunologically identical antigen was detected in adeno- and neurohypophysis. The physiological function of GP III is still unknown. It does not demonstrate any of the enzymatic activities so far known to occur in chromaffin granules.     

Subcellular Distribution of 65,000 Calmodulin-Binding Protein (p65) and Synaptophysin (p38) in Adrenal Medulla

Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.     

Dynamic changes in chromaffin cell cytoskeleton as prelude to exocytosis

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 α-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 Ca²⁺-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 pI_s, 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 μM) or high K+ (56 mM) produces a redistribution of subplasmalemmal scinderin and actin disassembly, which preceded exocytosis. The redistribution of scinderin and exocytosis is Ca²⁺-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-β-hydroxylase (anti-DβH) surface staining, are preferentially localized in plasma membrane areas devoid of F-actin.     

IMMUNOLOGICAL STUDIES ON A MEMBRANE PROTEIN (CHROMOMEMBRIN B) OF CATECHOLAMINE-STORING VESICLES

Abstract— Rabbits were immunized with chromomembrin B, i.e. a membrane protein isolated from chromaffin granules of bovine adrenal medulla. When the rabbit sera were tested by immunodiffusion in the presence of various detergents, only negative results were obtained, whereas with complement fixation antibodies could be demonstrated. With this method the subcellular distribution of chromomembrin B in bovine adrenal medulla was determined. The results demonstrate that this protein is specifically localized in the membranes of chromaffin granules. In the mitochondrial and microsomal fractions it is present only in small amounts which are attributable to a contamination of these fractions with chromaffin granules. The subcellular distribution of chromomembrin R in bovine splenic nerves indicates that this antigen is also found in the membranes of noradrenalinestoring vesicles of sympathetic nerve. Chromomembrin B or a related antigen was detected in chromaffin grades isolated from pig and rat adrenal and in those isolated from a human phaeochromocytoma. It is also present in total membranes obtained from posterior and anterior hypophysis, but it is absent from membranes isolated from parotid gland, liver and adrenal cortex. This paper illustrates how a membrane protein which requires detergents for its solubilization can be characterized and measured by immunological methods.     

Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling

The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.     

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.     

cis-Unsaturated fatty acids induce the fusion of chromaffin granules aggregated by synexin

When isolated chromaffin granules were aggregated by synexin (a Ca2+-binding protein present in chromaffin and other secretory tissues) and then exposed to cis-unsaturated fatty acids at 37 degrees C, they fused together to form large vesicles. The fusion was monitored by phase and electron microscopy and by turbidity measurements on the granule suspension. Arachidonic acid was the most effective fusogen, whereas trans-unsaturated fatty acids, saturated fatty acids, detergents or lysolecithin were inactive. During fusion some of the epinephrine of the granules was released but the soluble core proteins remained trapped in the resulting vesicles. These vesicles swelled to enclose the maximum volume. Although this swelling could be inhibited by increasing the osmotic strength of the medium, it did not appear to depend on the chemiosmotic properties of the granule membranes as it was not influenced by ATP, a proton ionophore, or an anion transport inhibitor. The regulators of this in vitro fusion--Ca2+, synexin, and free, cis-unsaturated fatty acids--may be present in the cytoplasm of the chromaffin cell when it is stimulated to release epinephrine and granule proteins by exocytosis. Therefore, this fusion event may be the same that occurs between chromaffin granules undergoing compound exocytosis.     

Adrenal paraneurone contractile proteins and stimulus-secretion coupling

Actin, myosin, and alpha-actinin have been isolated from adrenal chromaffin cells and characterized. Their physicochemical properties have been studied and their cell localization revealed by biochemical, immunocytochemical, and ultrastructural techniques. Alpha-actinin is a component of chromaffin granule membranes and some of the cell actin copurifies with these secretory granules. Myosin is not detected in the granules but is present mainly in the cytosol. Trifluoperazine, a calmodulin antagonist, blocks stimulation-induced hormone release from chromaffin cells at a step distal from Ca2+ entry. High affinity calmodulin binding sites have also been found in chromaffin granule membranes. Furthermore, microinjection of calmodulin antibodies into chromaffin cells blocks hormone output in response to stimulation. In view of the above findings, the possible roles of contractile proteins and calmodulin in chromaffin cell functions is discussed.