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Following incubation of human fibroblasts with Ca2+ ionophore A23187, we found strong immunofluorescence labelling of the nuclear envelope by annexin IV antibody. Using confocal imaging of cells loaded with Fluo-3, we showed that A23187 generates an intense and sustained rise of Ca2+ in the nucleus. By contrast, stimulation without extracellular Ca2+ produces only a brief rise in nuclear Ca2+ that does not promote annexin IV translocation to the nuclear envelope, and compounds that induce only a transient increase of nuclear Ca2+ do not support translocation of annexin IV. In addition, annexin V was also translocated to the nuclear envelope by A23187, but distribution of annexins I, II, VI and VII is unaffected. In in vitro assays with isolated nuclei, annexin V was also found to bind to the nuclear envelope in a Ca2+-dependent manner. These results demonstrate that the translocation to the nuclear envelope of different types of Ca2+-regulated proteins is directly triggered by a major rise of Ca2+ in the nucleus.

The annexin (Anx) gene family comprises a set of calcium-dependent membrane binding proteins, which have been implicated in a wide variety of cellular processes including membrane fusion and calcium channel activity. We report here that cAMP activates Ca2+-dependent aggregation of both phosphatidylserine (PS) liposomes and bovine chromaffin granules driven by [des 1–12]annexin I (lipocortin I, AnxI). The mechanism of cAMP action involves an increase in AnxI-dependent cooperativity on the rate of such a reaction without affecting the corresponding k1/2 values. Cyclic AMP causes the values of the Hill coefficient (nH) for AnxI to change from 3 to 6 in both PS liposomes and chromaffin granules. By contrast, ATP inhibits the rate of aggregation activity without affecting the cooperativity or the extent of aggregation process. We were also able to photolabel AnxI specifically with an 8-azido analogue of cAMP by a calcium-independent process. Such a process is saturable, yielding a Kd = 0.8 μM by Scatchard analysis. Specific displacement occurs in the presence of cAMP and ATP. Finally, we found that cAMP alters the conductance of calcium channels formed by AnxI in planar lipid bilayers. We interpret these data to indicate that AnxI binds both calcium and cAMP independently, and that both actions have functional consequences. This is the first report of a nucleotide binding function for a member of the annexin gene family.

Adrenal chromaffin cells have been found to accumulate ascorbic acid by a saturable high affinity mechanism that is inhibited by hydrocortisone. The rate of ascorbic acid transport into cells was linear for at least 1 h and had a Km of 103 microM, a value approaching the reported concentration of ascorbic acid in the adrenal vein during stress. The uptake process itself, representing net accumulation rather than exchange, was inhibited by 0 degrees C, lack of sodium, ouabain, and by dinitrophenol and iodoacetate. Hydrocortisone but not the inactive analogue hydrocortisone hemisuccinate was found to inhibit ascorbic acid uptake in a reversible manner, with an ID50 of 62 microM. This value was within the reported steroid concentration in the adrenal portal system during a significant stress. Both ascorbic acid and hydrocortisone are secreted from cortical cells during stress into the adrenal portal system and thus contact medullary chromaffin cells. We suggest that the control of ascorbic acid uptake by hydrocortisone indicates the existence of a heretofore unanticipated biochemical aspect of the adrenal stress response.

A cDNA containing the entire coding region for human nucleolin has been isolated from a λ gt10 human retinal library using a bovine cDNA probe. The cDNA hybridized to a transcript of 3000 bases from fast-dividing cells, as well as terminally differentiated tissues of several species. Translation of the nucleotide sequence revealed a long open reading frame which predicts a 707 amino acid protein with several distinct domains. These include repeating elements, four conserved RNA-binding regions, a glycine-rich carboxy-terminal domain and sites for phosphorylation, glycosylation and dibasic cleavage. Human and bovine nucleolin exhibited more additions and/or substitutions of aspartate, glutamate and serine residues in the chromatin-binding domains by comparison with the hamster and mouse nucleolins. These differences may be related to species-specific functions in transcription.

The influence of cytoskeletal elements on the chromaffm granule function was studied using a model system consisting of purified granule membranes and F-actin. The membrane ATPase was partially inactivated by incubation at 37 °C, and this inactivation was prevented by adding F-actin. The stabilizing action of F-actin on the ATPase was abolished by adding DNase I. Detergent-solubilized ATPase was more rapidly and profoundly inactivated, but was not stabilized by F-actin. The stabilization of ATPase by F-actin may be due to the cross-linking of granule membranes with F-actin and the native structure of the granule membrane may be required for preserving the stability of membrane ATPase. These findings thus suggest the possibility that the interaction of microfilaments with chromaffin granules may influence the function of chromaffin granules within the cell.

We report the isolation and purification of synhibin, a new M r 68000 protein, which inhibits synexin. Synexin mediates Ca 2+ -dependent chromaffin granule aggregation and fusion, processes perhaps important during exocytosis. Our data indicate that synhibin action involves competition with synexin for a site on the chromaffin granule membrane involved in membrane contact. Synhibin may thus be an important intracellular regulator of synexin action during secretion.

Pertussis toxin was found to stimulate catecholamine release from bovine adrenal chromaffin cells in a Ca2+ -dependent manner and in the absence of any stimulatory or inhibitory agonists for this cell. The release of catecholamine was associated with the ADP-ribosylation of an approx. 40 kDa protein present in the total membrane fraction. These results are consistent with the existence of an exocytosis-linked G-protein.

Oriented plasma membrane fragments from chromaffin cells, isolated on polylysine-coated polyacrylamide beads, bind synexin in a calcium dependent manner. Synexin binding was also detected on beads coated with chromaffin granule membranes, but not to beads coated with erythrocyte membranes or to uncoated beads. Synexin binding to plasma membrane coated beads showed a specific requirement for calcium (K12 = 200 μM) and was insensitive to other divalent cations such as magnesium, strontium and barium. Synexin binding to either plasma membrane or granule membrane coated beads was saturable, was partially reversible by EGTA and was directly observed by SDS-polyacrylamide gel electrophoresis.

The phorbol ester, 4β-phorbol 12-myristate acetate (TPA), increased the extent of catecholamine release induced by Ca2+, without affecting the basal release response in digitonin-permeabilized chromaffin cells. This finding is consistent with the hypothesis that protein kinase C has a role to play in stimulus-secretion coupling in the bovine adrenal medullary chromaffin cell. Phorbol esterCathecholamineProtein kinase CChromaffinSecretionCa2+-induced release

The luminescent oxidation of luciferin has been used to monitor acetylcholine-induced ATP release from cultured bovine chromaffin cells. Acetylcholine (1-100 μM) evoked ATP release of up to 30% of the total cellular ATP. This secretion required external free calcium and could also be elicited by K+-induced membrane depolarization. The size of the cytosolic ATP compartment was estimated as 5% of the ATP in the cell by solubilising the cell membrane using digitonin (20 μM) or by application to the cells of brief pulses (2 μs) of high electric field (2000 V cm ). Blockers of the voltage-gated Ca 2+ channel effectively blocked K + -induced ATP release, while the acetylcholine antagonists d-tubocurarine and β-bungarotoxin inhibited the acetylcholine-induced release of ATP. These data support the concept that ATP is released together with the catecholamines by exocytosis of chromaffin granule contents. ATP secretion Chromaffin cell Acetylcholine channel Exocytosis Catecholamine