Cytoskeletal control of vesicle transport and exocytosis in chromaffin cells.

Chromaffin cell exocytosis is a fascinating interplay between secretory vesicles and cellular components. One of these components is the cytoskeleton and its associated regulatory proteins. Transport of chromaffin secretory granules from their site of biosynthesis towards the active site of exocytosis requires both F-actin fine remodelling as well as microtubule trails. At least two molecular motors, myosins II and V, seem to play a crucial role in the control of F-actin dynamics and vectorial vesicle displacement respectively. Vesicle movement experiences spatial restrictions as they approach the cell cortical region, where the F-actin meshwork constitutes a barrier-limiting vesicle access to the plasmalemma. During secretion, cortical F-actin is locally disrupted providing access of vesicles to release sites on the plasmalemma. Removal of the stimulus restores cortical F-actin. Two pathways (Ca2+-scinderin and PKC-MARCKS) control F-actin changes during the secretory cycle . Furthermore, GTPases such as RhoA, that controls F-actin network integrity, and Cdc42 signalling which induces the formation of local actin filaments at active sites, provide additional evidence on the importance of F-actin as a key element in vesicle transport and in the exocytotic machinery of chromaffin cells.

Mitochondrial calcium sequestration and protein kinase C cooperate in the regulation of cortical F-actin disassembly and secretion in bovine chromaffin cells.

Mitochondria play an important role in the homeostasis of intracellular Ca(2+) and regulate its availability for exocytosis. Inhibitors of mitochondria Ca(2+) uptake such as protonophore CCCP potentiate the secretory response to a depolarizing pulse of K(+). Exposure of cells to agents that directly (cytochalasin D, latrunculin B) or indirectly (PMA) disrupt cortical F-actin networks also potentiate the secretory response to high K(+). The effects of cytochalasin D and CCCP on secretion were additive whereas those of PMA and CCCP were not; this suggests different mechanisms for cytochalasin D and CCCP and a similar mechanism for PMA and CCCP. Mitochondria were the site of action of CCCP, because the potentiation of secretion by CCCP was observed even after depletion of Ca(2+) from the endoplasmic reticulum. CCCP induced a small increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that was not modified by the protein kinase C (PKC) inhibitor chelerythrine. Both CCCP and PMA induced cortical F-actin disassembly, an effect abolished by chelerythrine. In addition, rotenone and oligomycin A, two other mitochondrial inhibitors, also evoked cortical F-actin disassembly and potentiated secretion; again, these effects were blocked by chelerythrine. CCCP also enhanced the phosphorylation of PKC and myristoylated alanine-rich C kinase substance (MARCKS), and these were also inhibited by chelerythrine. The results suggest that the rapid sequestration of Ca(2+) by mitochondria would protect the cell from an enhanced PKC activation and cortical F-actin disassembly, thereby limiting the magnitude of the secretory response.

In vivo erythroid recovery following paclitaxel injury: correlation between GATA-1, c-MYB, NF-E2, Epo receptor expressions, and apoptosis.

Paclitaxel (Px) is a cancer chemotherapeutic agent that causes bone marrow (BM) cytotoxicity by microtubule stabilization and by modifications in the expression of several genes. Hematopoietic progenitors show severe alterations following Px injury. Erythropoietic recovery should be accompanied by changes in the expression of transcription factors such as c-MYB, GATA-1, NF-E2, Bcl-x(L), and erythropoietin receptor (Epo-R). The aim of this work was to study the in vivo recovery of erythropoiesis and to correlate transcription factors, Bcl-x(L), and Epo-R expressions to apoptosis and changes in proliferation of murine erythroid progenitors following a single dose of Px (29 mg/kg, i.p.). BM total and differential cellularities, apoptosis (TdT-mediated dUTP Nick-End Labeling [TUNEL] assay), clonogenic assays, and immunoblots for transcription factors, Epo-R, and Bcl-x(L) were performed each day for 5 days post-injury. Apoptosis (24 +/- 0.81%, P < 0.01), inhibition of colony growth (burst-forming units-erythroid [BFU-E] and granulocyte-erythroid-macrophage [GEM]), and decrease in BM cellularities (28 +/- 4.2% of control) were maximal at 24 h following Px. The highest apoptosis was concomitant with the lowest BM cellularities. Apoptosis returned to normal values (3.08 +/- 0.61%) by day 3 post-Px. Up-regulation of c-MYB, GATA-1, Epo-R, and Bcl-x(L) expressions were observed between 24 and 48 h following Px. Correlations among c-MYB, GATA-1, Bcl-x(L), and Epo-R were extremely significant. Maximal expression of NF-E2 was observed on day 3 concomitant with the rise (threefold) of early erythroid precursors (BFU-E). Thus, cells that survive injury seem to be stimulated to produce early (24-48 h) erythroid-related and antiapoptotic proteins. Therefore, the results suggest an in vivo interplay between specific transcription factors and Bcl-x(L) during progenitor cell survival and proliferation; mechanisms triggered to restore size and composition of the erythroid compartment.

The role of different Scinderin domains in the control of F-actin cytoskeleton during exocytosis.

Scinderin (Sc), a Ca(2+)-regulated actin-binding protein, has been previously shown to control submembranous actin dynamics in regulated secretion. The results of the present study suggest the possibility that Sc might act as a molecular switch in the control of cortical F-actin dynamics during secretion.

Change of quantal size and parameters of release with stimulation in bovine chromaffin cells.

Changes in quantal size and in the parameters of release were examined in chromaffin cells using amperometric recordings during and following various stimuli that induce secretion. As a general rule, a greater quantal content was associated with a greater quantal size. Following a short depolarizing pulse (0.5-2 s; 100 mV from a holding potential of -80 mV), the mean value of quantal size increased by 54% over several seconds before gradually (over tens of seconds) returning toward the control value, whilst its variability rose by 62%. The changes observed following 30-s applications of high extracellular K+ (50 mM) were more modest. A rapid application of short depolarizing pulses (2 s every 10-20 s; 100 mV from a holding potential of -80 mV) also led, at least initially, to greater quantal content and quantal size. Mean quantal size rose initially by 68%, but decreased subsequently to 31% below pre-stimulation levels. In whole-clamped cells, the frequency of quantal release can rise abruptly, probably owing to a mechanical disturbance that makes the membrane leaky to Ca2+. In such cases, a marked rise in quantal content (>ten-fold) was paralleled by an almost as dramatic (up to ten-fold) rise in quantal size and an important, although less pronounced and slower, rise in its variability (up to four-fold). The return toward control values of mean quantal size occurred over several minutes, whilst its variability decayed more slowly. The release parameters were evaluated directly from the number of events to avoid a large and time-dependent contribution of the amplitude variability of spontaneous amperometric current spikes (minis). In general, the greater probability of release contributed more than the greater size of the immediately available store to the increase in quantal output. In conclusion, quantal size was found to be highly labile. Its change can alter strongly the facilitation and depression of evoked quantal output and probably occurs due to a preferential release of large vesicles that are more efficient barriers to Ca2+ diffusion when Ca2+ rises rapidly following a synchronous opening of several Ca2+ channels. When intracellular Ca2+ levels rise slowly to threshold levels for secretion, as during an asynchronous and generally spontaneous release, vesicles are less effective diffusion barriers and quantal size changes less.

Pathways that control cortical F-actin dynamics during secretion.

Chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane which acts as a barrier to the chromaffin vesicles access to exocytotic sites. Disassembly of cortical F-actin in response to stimulation allows the movement of vesicles from the reserve pool to the release-ready vesicle pool and, therefore, to exocytotic sites. The dynamics of cortical F-actin is controlled by two mechanisms: a) stimulation-induced Ca2+ entry and scinderin activation and b) protein kinase C (PKC) activation and MARCKS phosphorylation as demonstrated here by experiments with recombinant proteins, antisense olygodeoxynucleotides and vector mediated transient expressions. Under physiological conditions (i.e., cholinergic receptor stimulation followed by Ca2+ entry), mechanism (a) is the most important for the control of cortical F-actin network whereas when Ca2+ is released from intracellular stores (i.e., histamine stimulation) cortical F-actin is regulated mainly by mechanism b.

Expression of scinderin in megakaryoblastic leukemia cells induces differentiation, maturation, and apoptosis with release of plateletlike particles and inhibits proliferation and tumorigenesis.

Rapid proliferation of atypical megakaryoblasts is a characteristic of megakaryoblastic leukemia. Cells from patients with this disorder and cell lines established from this type of leukemia showed the presence of gelsolin but the absence of scinderin expression, 2 filamentous actin-severing proteins present in normal megakaryocytes and platelets. Vector-mediated expression of scinderin in the megakaryoblastic cell line MEG-01 induced a decrease in both F-actin and gelsolin. This was accompanied by increased Rac2 expression and by activation of the PAK/MEKK.SEK/JNK/c-jun, c-fos transduction pathway. The Raf/MEK/ERK pathway was also activated in these cells. Transduction pathway activation was followed by cell differentiation, polyploidization, maturation, and apoptosis with release of platelet-like particles. Particles expressed surface CD41a antigen (glycoprotein IIb/IIIa or fibrinogen receptor), had dense bodies, high-affinity serotonin transport, and circular array of microtubules. Treatment of particles with thrombin induced serotonin release and aggregation that was blocked by CD41a antibodies. PAC-1 antibodies also blocked aggregation. Exposure of cells to PD98059, a blocker of MEK, inhibited antigen CD41a expression, increases in cell volume, and number of protoplasmic extensions. Cell proliferation and cell ability to form tumors in nude mice were also inhibited by the expression of scinderin. MEG-01 cells expressing scinderin had the same fate in vivo as in culture. Thus, when injected into nude mice, they entered apoptosis and released platelet-like particles. The lack of scinderin expression in megakaryoblastic leukemia cells seems to be responsible for their inability to enter into differentiation and maturation pathways characteristic of their normal counterparts.

Chromaffin cell F-actin disassembly and potentiation of catecholamine release in response to protein kinase C activation by phorbol esters is mediated through myristoylated alanine-rich C kinase substrate phosphorylation.

The large majority of chromaffin vesicles are excluded from the plasma membrane by a cortical F-actin network. Treatment of chromaffin cells with phorbol 12-myristate 13-acetate produces disassembly of cortical F-actin, increasing the number of vesicles at release sites (Vitale, M. L., Seward, E. P., and Trifaró, J. M. (1995) Neuron 14, 353-363). Here, we provide evidence for involvement of myristoylated alanine-rich protein kinase C substrate (MARCKS), a protein kinase C substrate, in chromaffin cell secretion. MARCKS binds and cross-links F-actin, the latter is inhibited by protein kinase C-induced MARCKS phosphorylation. MARCKS was found in chromaffin cells by immunoblotting. MARCKS was also detected by immunoprecipitation. In intact or permeabilized cells MARCKS phosphorylation increased upon stimulation with 10(-7) m phorbol 12-myristate 13-acetate. This was accompanied by cortical F-actin disassembly and potentiation of secretion. MARCKS phosphorylation, cortical F-actin disassembly, and potentiation of Ca(2+)-evoked secretion were inhibited by a peptide (MARCKS phosphorylation site domain sequence (MPSD)) with amino acid sequence corresponding to MARCKS phosphorylation site. MPSD was phosphorylated in the process. A similar peptide (alanine-substituted phosphorylated site domain) with four serine residues of MPSD substituted by alanines was ineffective. These results provide the first evidence for MARCKS involvement in chromaffin cell secretion and suggest that regulation of cortical F-actin cross-linking might be involved in this process.

Myristoylated alanine-rich C kinase substrate phosphorylation is involved in thrombin-induced serotonin release from platelets.

Stimulation of platelets by thrombin induces protein kinase C (PKC) activation, phosphorylation of pleckstrin, aggregation and serotonin release. Here, we demonstrate that, in human platelets, thrombin stimulation also induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) and serotonin release in intact and digitonin-permeabilized platelets. MARCKS is known to bind actin and cross-link actin filaments, and this is inhibited by PKC-evoked MARCKS phosphorylation. MARCKS phosphorylation and serotonin release in response to increasing concentrations of thrombin have a similar EC50 and time course and, in permeabilized platelets, peptide MPSD, with an amino acid sequence corresponding to the phosphorylation site domain of MARCKS, blocked both responses. However, pleckstrin and myosin light chain phosphorylations were not modified. Ala-MPSD, in which the four serine residues of MPSD were substituted by alanines was ineffective. The results suggest a role for MARCKS in platelet secretion. The fact that pleckstrin phosphorylation has a different time course and was not modified in the presence of MPSD when MARCKS phosphorylation and serotonin release were inhibited would suggest either that pleckstrin phosphorylation is unrelated to secretion or that it might only be involved upstream in the events leading to secretion.

An antisense oligodeoxynucleotide targeted to chromaffin cell scinderin gene decreased scinderin levels and inhibited depolarization-induced cortical F-actin disassembly and exocytosis.

Chromaffin cell secretion requires cortical F-actin disassembly and it has been suggested that scinderin, a Ca2+ dependent F-actin severing protein, controls cortical actin dynamics. An antisense oligodeoxynucleotide targeting the scinderin gene was used to decrease the expression of the protein and access its role in secretion. Treatment with 2 microM scinderin antisense oligodeoxynucleotide for 4 days produced a significant decrease in scinderin expression and its mRNA levels. The expression of gelsolin, another F-actin severing protein, was not affected. Scinderin decrease was accompanied by concomitant and parallel decreases in depolarization-evoked cortical F-actin disassembly and exocytosis. Similar treatment with a mismatched oligodeoxynucleotide produced no effects. Scinderin antisense oligodeoxynucleotide treatment was also a very effective inhibitor of exocytosis in digitonin-permeabilized cells stimulated with increasing concentrations of Ca2+. This ruled out scinderin antisense interference with stimulation-induced depolarization or Ca2+ channel activation. Scinderin antisense treatment decreased the maximum (B(max)) secretory response to Ca2+ without modifying the affinity (K(m)) of the cation for the exocytotic machinery. Moreover, the antisense treatment did not affect norepinephrine uptake or the expression of dopamine ss-hydroxylase, suggesting that the number and function of chromaffin vesicles was not modified. In addition, scinderin antisense treatment did not alter the expression of proteins involved in vesicle-plasma membrane fusion, such as synaptophysin, synaptotagmin or syntaxin, indicating a lack of effects on the fusion machinery components. These observations strongly suggest that scinderin is a key player in the events involved in the secretory process.

Scinderin, a Ca2+-dependent actin filament severing protein that controls cortical actin network dynamics during secretion.

Secretory vesicles are localized in specific compartments within neurosecretory cells. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is controlled and regulated by Ca2+, scinderin and the cortical F-actin network. Cortical F-actin disassembly is produced by the filament severing activity of scinderin. This Ca2+-dependent activity of scinderin together with its Ca2+-independent actin nucleating activity, control cortical F-actin dynamics during the secretory cycle. A good understanding of the interaction of actin with scinderin and of the role of this protein in secretion has been provided by the analysis of the molecular structure of scinderin together with the use of recombinant proteins corresponding to its different domains.

Platelet secretion induced by phorbol esters stimulation is mediated through phosphorylation of MARCKS: a MARCKS-derived peptide blocks MARCKS phosphorylation and serotonin release without affecting pleckstrin phosphorylation.

Previous experiments suggest that actin disassembly, perhaps at a specific site, is required for platelet secretion. Platelet stimulation by phorbol 12-myristate 13-acetate (PMA) induced pleckstrin phosphorylation, platelet aggregation, and secretion. Inhibition of protein kinase C (PKC) is accompanied by inhibition of pleckstrin phosphorylation and serotonin secretion. Here, we demonstrate the presence of myristoylated alanine-rich C kinase substrate (MARCKS), another PKC substrate, in platelets and its phosphorylation during PMA stimulation. MARCKS is known to bind actin and to cross-link actin filaments; the latter is inhibited by PKC-induced MARCKS phosphorylation. MARCKS phosphorylation and serotonin release from permeabilized platelets have the same time course and were blocked by a peptide (MPSD) with the amino acid sequence corresponding to the phosphorylation site domain of MARCKS. Pleckstrin and myosin light chain phosphorylation was not modified. A peptide (Ala-MPSD) in which the four serine residues of MPSD were substituted by alanines was ineffective. These results provide the first evidence that MARCKS may play a role in platelet secretion. Moreover, pleckstrin phosphorylation has a different time course than that of MARCKS or serotonin release and was not modified when MARCKS phosphorylation and serotonin release were inhibited, suggesting that pleckstrin is either not directly involved in secretion or that it might only be involved upstream in the cascade of events leading to exocytosis.

Scinderin and cortical F-actin are components of the secretory machinery.

Secretory vesicle exocytosis is the mechanism of release of neurotransmitters and neuropeptides. Secretory vesicles are localized in at least two morphologically and functionally distinct compartments: the reserve pool and the release-ready pool. Filamentous actin networks play an important role in this compartmentalization and in the trafficking of vesicles between these compartments. The cortical F-actin network constitutes a barrier (negative clamp) to the movement of secretory vesicles to release sites, and it must be locally disassembled to allow translocation of secretory vesicles in preparation for exocytosis. The disassembly of the cortical F-actin network is controlled by scinderin (a Ca(2+)-dependent F-actin severing protein) upon activation by Ca2+ entering the cells during stimulation. There are several factors that regulate scinderin activation (i.e., Ca2+ levels, phosphatidylinositol 4,5-bisphosphate (PIP2), etc.). The results suggest that scinderin and the cortical F-actin network are components of the secretory machinery.

Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa.

We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.

Comparison of vesicular volume and quantal size in bovine chromaffin cells.

Electrochemical measurements of vesicular content released were compared with the morphometric measurements of vesicular size in bovine chromaffin cells. Cross-sectional vesicular diameters were determined from electron micrographs. Two methods were used to determine the frequency histograms of "true" vesicular diameters (i.e. diameters of the vesicles in the equatorial plane): (i) "peeling off" method [Coupland R. E. (1968), Nature 217, 384-388], and (ii) summation of individual probabilities of "true" vesicular diameters. Quantal size was estimated from the area under the spontaneous current spike detected electrochemically. The frequency histograms of "true" vesicular diameters are found to be skewed (thus not well described by a Gaussian function) irrespective of the method used to calculate them, as are the frequency histograms of the cube roots of the quantal sizes. Furthermore, we also find that the frequency histograms of electrochemical measurements (the cube roots of quantal sizes) have lower skews and coefficients of variation than those of morphometric measurements ("true" vesicular diameters), with discrepancy being especially pronounced for noradrenaline-secreting cells. Such a difference in both coefficients of variation and skews suggests that the intravesicular catecholamine concentration is not uniform, but that it is lower for vesicles of larger size. In conclusion a variety of factors--vesicular volume, vesicular surface area to volume ratio, binding capacity of chromogranin and/or ATP, likely determines the amount of catecholamine stored in the vesicle.

Light and electron microscopic study of changes in the organization of the cortical actin cytoskeleton during chromaffin cell secretion.

Chromaffin cells cultured for 2 days were incubated in the absence or presence of 10 microM nicotine for 40 sec. Resting and stimulated cells were fixed and either prepared for fluorescence microscopy or treated with Triton X-100 to obtain cytoskeletons for ultrastructural studies. Electron microscopy of cytoskeletons revealed the presence of polygonal areas devoid of actin filaments only in nicotinic receptor-stimulated cells. Staining of these cytoskeleton preparations with rhodamine-phalloidin, a probe for filamentous actin, produced fluorescent patterns and three-dimensional images similar to those obtained from resting or stimulated intact cells prepared directly for fluorescence microscopy. Moreover, the percentage of stimulated cells showing disrupted cytoskeleton at the electron microscopic level was similar to the percentage of stimulated cells showing patched rhodamine fluorescence at the fluorescence microscopic level. In addition, cells stimulated with nicotine for 40 sec showed a fivefold increase in amine output and a significant decrease in F-actin levels. These results provide the first ultrastructural evidence for nicotinic receptor-evoked chromaffin cell F-actin disassembly and show that the rhodamine-phalloidin-unstained areas observed in fluorescence microscopy represent the areas devoid of filamentous actin observed at the electron microscopic level.

Localization by segmental deletion analysis and functional characterization of a third actin-binding site in domain 5 of scinderin.

Scinderin is a Ca2+-dependent actin filament severing protein present in a variety of secretory cells. Previous work suggests that scinderin-evoked cortical F-actin disassembly is required for secretion because local disassembly of cortical cytoskeleton allows secretory vesicle exocytosis (Vitale, M. L., Rodríguez Del Castillo, A., Tchakarov, L., and Trifaró, J.-M. (1991) J. Cell Biol. 113, 1057-1067). Scinderin has six domains each containing three internal sequence motifs, two actin, and two phosphatidylinositol disphosphate-binding sites in domains 1 and 2. In this paper we report the presence of another actin-binding site at the NH2-terminal of domain 5 (Sc511-518). This site binds actin in a Ca2+-independent manner and a recombinant fragment (Sc5-6 or Sc502-715) containing this site binds to actin-DNase-I-Sepharose 4B beads, co-sediments with actin and is able to nucleate actin assembly. Recombinant ScL5-6, a fusion protein devoid of the actin-binding site (Sc519-715), did not exhibit these properties. Moreover, Sc-ABP3, a peptide constructed with sequence (RLFQVRRNLASIT) identical to Sc511-523 blocked the binding of Sc5-6 to actin. Sc5-6 and Sc-ABP3 also prevented the actin severing activity of recombinant full-length scinderin (r-Sc) and inhibited the potentiation by r-Sc of Ca2+-evoked release of serotonin from permeabilized platelets. On the other hand, ScL5-6 failed to block the effect of r-Sc on platelet serotonin release. Sc1-4,6, a construct devoid of domain 5, was able to sever but unable to nucleate actin, indicating that an actin nucleation site of scinderin was in domain 5. The results suggest that scinderin, in addition to binding actin on sites present in domains 1 and 2, must bind actin on a third site in domain 5 to sever and nucleate actin effectively.

Secretory vesicle pools and rate and kinetics of single vesicle exocytosis in neurosecretory cells.

Secretory vesicles are localized in specific compartments within neurosecretory cells. Morphometric, cytochemical and electrophysiological techniques have allowed the definition of secretory vesicle compartments. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is not random, but an event controlled and regulated by Ca2+ and the cortical F-actin network. Cortical F-actin disassembly, a Ca(2+)-dependent event, controls the transit of secretory vesicles from the reserve compartment to the release-ready vesicle pool. Furthermore, the recent development of new technical approaches (patch-clamp membrane capacitance, electrochemical detection of amines with carbon-fibre microelectrodes) has now permitted us to understand the kinetics of single vesicle exocytosis.

Differential effects of forskolin and 1,9-dideoxy-forskolin on nicotinic receptor- and K+-induced responses in chromaffin cells.

The diterpene forskolin inhibits nicotine-evoked chromaffin cell Ca2+ influx, scinderin redistribution, F-actin disassembly and catecholamine secretion in a concentration-dependent (10-50 microM) fashion. On the other hand, forskolin showed weak inhibitory effects when the same responses were elicited by K+-induced depolarization. Similar concentrations of 1,9-dideoxy-forskolin, a forskolin analog which does not activate adenylate cyclase, blocked very effectively the responses evoked by either of the two stimuli. Patch-clamp (whole-cell configuration) studies demonstrated that both diterpenes blocked fast and reversibly peak and total chromaffin cell nicotinic acetylcholine receptor currents, effects not mediated through adenylate cyclase activation. Moreover, both forskolin and 1,9-dideoxy-forskolin exhibited Ca2+ channel blocking properties. However, 1,9-dideoxy-forskolin was more potent than forskolin as a Ca2+ channel blocker. Furthermore, 1,9-dideoxy-forskolin was also more potent than forskolin as a nicotinic acetylcholine receptor and Ca2+ channel blocker and it was more potent as a nicotinic acetylcholine receptor blocker than Ca2+ channel blocker. The results showed powerful cAMP-independent effects of the diterpenes and suggest caution in interpretation of cAMP effects on chromaffin cells when its cellular levels are modified by forskolin.

Recombinant scinderin enhances exocytosis, an effect blocked by two scinderin-derived actin-binding peptides and PIP2.

The cortical F-actin cytoskeleton represents a negative control for secretion, and it must be locally disassembled to allow chromaffin vesicle exocytosis. Recombinant scinderin (a Ca(2+)-dependent F-actin-severing protein) potentiated Ca(2+)-evoked F-actin disassembly and exocytosis in permeabilized chromaffin cells, an effect blocked by peptides Sc-ABP1 and Sc-ABP2 (with sequences corresponding to two actin-binding sites of scinderin), exogenous gamma-actin, or phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 effect was blocked by peptide Sc-PIP2BP (with sequence corresponding to a PIP2-binding site of scinderin). Truncated scinderin254-715 (lacking actin-severing domains) did not potentiate exocytosis. Sc-ABP1, Sc-ABP2, and gamma-actin also inhibited exocytosis in the absence of recombinant scinderin, suggesting an inhibition of endogenous scinderin. Results suggest that scinderin-evoked cortical F-actin disassembly is required for secretion and that scinderin is an important component of the exocytotic machinery.