The control of cytoskeletal actin and exocytosis in intact and permeabilized adrenal chromaffin cells: role of calcium and protein kinase C

The control of cytoskeletal actin and exocytosis was examined in intact and digitonin-permeabilized chromaffin cells. Cytoskeletal actin was assayed by determining the actin content of Triton-insoluble cytoskeletons. The secretagogues nicotine, high K+ and Ba2+ resulted in a rapid reduction in the amount of actin associated with the cytoskeleton. The effect of nicotine but not high K+ on cytoskeletal actin was independent of external Ca2+ and the reduction in cytoskeletal actin was mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate suggesting a role for protein kinase C. In digitonin-permeabilized cells micromolar calcium produced both catecholamine secretion and a reduction in cytoskeletal actin. The reduction in cytoskeletal actin was transient. Secretion was enhanced by the GTP analogue guanosine 5'-(3-O-thio)triphosphate and the analogue also reduced cytoskeletal actin at low calcium levels. The effects of guanosine 5'-(3-O-thio)triphosphate were inhibited by the phospholipase C inhibitor neomycin and were mimicked by 12-O-tetradecanoylphorbol-13-acetate. An additional GTP analogue, guanyl-5'-yl imidodiphosphate, had no effect on cytoskeletal actin. These results provide further evidence for a requirement for reorganisation of cortical actin in the secretory processes and suggest that the reduction in actin associated with the cytoskeleton may be mediated by protein kinase C and/or calcium in intact and permeabilized chromaffin cells.     

Kinetics of exocytosis in adrenal chromaffin cells

Upon repetitive or maintained stimulation, chromaffin cells secrete catecholamines initially at a very high rate which then relaxes with multiple kinetic components. The complex kinetics are often modeled as resulting from the successive depletion of several functional pools of secretory granules which may reflect specific protein-mediated steps in granule maturation. The fastest component represents granules fully primed for exocytosis. This 'readily releasable pool' may, under some circumstances, consist of only about a dozen granules which can be released within tens of milliseconds. Modulating the size of this pool may be an important way for cells to regulate secretion.     

Calcium gradients and exocytosis in bovine adrenal chromaffin cells

The relationship between the localized Ca(2+) concentration and depolarization-induced exocytosis was studied in patch-clamped adrenal chromaffin cells using pulsed-laser Ca(2+) imaging and membrane capacitance measurements. Short depolarizing voltage steps induced Ca(2+) gradients and small "synchronous" increases in capacitance during the pulses. Longer pulses increased the capacitance changes, which saturated at 16 fF, suggesting the presence of a small immediately releasable pool of fusion-ready vesicles. A Hill plot of the capacitance changes versus the estimated Ca(2+) concentration in a thin (100 nm) shell beneath the membrane gave n = 2.3 and K(d) = 1.4 microM. Repetitive stimulation elicited a more complex pattern of exocytosis: early pulses induced synchronous capacitance increases, but after five or more pulses there was facilitation of the synchronous responses and gradual increases in capacitance continued between pulses (asynchronous exocytosis) as the steep submembrane Ca(2+) gradients collapsed. Raising the pipette Ca(2+) concentration led to early facilitation of the synchronous response and early appearance of asynchronous exocytosis. We used this data to develop a kinetic model of depolarization-induced exocytosis, where Ca(2+)-dependent fusion of vesicles occurs from a small immediately releasable pool with an affinity of 1-2 microM and vesicles are mobilized to this pool in a Ca(2+)-dependent manner.     

Isoform-specific inhibition of voltage-sensitive Ca(2+) channels by protein kinase C in adrenal chromaffin cells

Selective protein kinase C (PKC) activators and inhibitors were used to investigate the involvement of specific PKC isoforms in the modulation of voltage-sensitive Ca(2+) channels (VSCCs) in bovine adrenal chromaffin cells. Exposure to the phorbol ester phorbol-12,13-dibutyrate (PDBu) inhibited the Ca(2+) currents elicited by depolarizing voltage steps. This inhibition was occluded by the PKC-specific inhibitor Ro 31-8220 but remained unaffected by G? 6976, a selective inhibitor of conventional PKC isoforms. PDBu treatment caused the translocation of PKC-alpha and -epsilon isoforms from cytosol to membranes. PKC-iota and -zeta showed no signs of translocation. It is concluded that VSCCs are specifically inhibited by the activation of PKC-epsilon in chromaffin cells. This may be relevant to the action of phospholipase-linked receptors involved in the control of Ca(2+) influx, both in catecholaminergic cells and other cell types.     

Correlation between vesicle quantal size and fusion pore release in chromaffin cell exocytosis

A significant number of exocytosis events recorded with amperometry demonstrate a prespike feature termed a "foot" and this foot has been correlated with messengers released via a transitory fusion pore before full exocytosis. We have compared amperometric spikes with a foot with spikes without a foot at chromaffin cells and found that the probability of detecting a distinct foot event is correlated to the amount of catecholamine released. The mean charge of the spikes with a foot was found to be twice that of the spikes without a foot, and the frequency of spikes displaying a foot was zero for small spikes increasing to approximately 50% for large spikes. It is hypothesized that in chromaffin cells, where the dense core is believed to nearly fill the vesicle, the expanding core is a controlling factor in opening the fusion pore, that prefusion of two smaller vesicles leads to excess membrane, and that this slows pore expansion leading to an increased observation of events with a foot. Clearly, the physicochemical properties of vesicles are key factors in the control of the dynamics of release through the fusion pore and the high and variable frequency of this release makes it highly significant.     

Regulation of exocytosis in adrenal chromaffin cells: focus on ARF and Rho GTPases

Neurons and neuroendocrine cells release transmitters and hormones by exocytosis, a highly regulated process in which secretory vesicles or granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Several stages have been recognized in exocytosis. After recruitment and docking at the plasma membrane, vesicles/granules enter a priming step, which is then followed by the fusion process. Cortical actin remodelling accompanies the exocytotic reaction, but the links between actin dynamics and trafficking events remain poorly understood. Here, we review the action of Rho and ADP-ribosylation factor (ARF) GTPases within the exocytotic pathway in adrenal chromaffin cells. Rho proteins are well known for their pivotal role in regulating the actin cytoskeleton. ARFs were originally identified as regulators of vesicle transport within cells. The possible interplay between these two families of GTPases and their downstream effectors provides novel insights into the mechanisms that govern exocytosis.     

Requirement for metalloendoprotease in exocytosis: evidence in mast cells and adrenal chromaffin cells

Exocytosis is initiated by the receptor-mediated influx of calcium that results in fusion of the secretory vesicle with the plasma membrane. We examined the possibility that calcium-dependent exocytosis in mast cells and adrenal chromaffin cells requires metalloendoprotease activity. Metalloendoprotease inhibitors and dipeptide substrates block exocytosis in these cells with the same specificity and dose dependency as that with which they interact with metalloendoproteases. Metalloendoprotease activity is identified in these cells with fluorogenic synthetic substrates, which also blocked exocytosis. Metalloendoprotease activity is highest in the plasma membrane of chromaffin cells. The metalloendoprotease appears to be required in exocytosis at a step dependent on or after calcium entry, since exocytosis initiated by direct calcium introduction in both mast cells and chromaffin cells is blocked by metalloendoprotease inhibitors.     

Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis

Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.     

Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine

We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.     

Regulation of the fusion pore conductance during exocytosis by cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase involved in synaptogenesis and brain development, and its enzymatic activity is essential for slow forms of synaptic vesicle endocytosis. Recent work also has implicated Cdk5 in exocytosis and synaptic plasticity. Pharmacological inhibition of Cdk5 modifies secretion in neuroendocrine cells, synaptosomes, and brain slices; however, the specific mechanisms involved remain unclear. Here we demonstrate that dominant-negative inhibition of Cdk5 increases quantal size and broadens the kinetics of individual exocytotic events measured by amperometry in adrenal chromaffin cells. Conversely, Cdk5 overexpression narrows the kinetics of fusion, consistent with an increase in the extent of kiss-and-run exocytosis. Cdk5 inhibition also increases the total charge and current of catecholamine released during the amperometric foot, representing a modification of the conductance of the initial fusion pore connecting the granule and plasma membrane. We suggest that these effects are not attributable to an alteration in catecholamine content of secretory granules and therefore represent an effect on the fusion mechanism itself. Finally, mutational silencing of the Cdk5 phosphorylation site in Munc18, an essential protein of the late stages of vesicle fusion, has identical effects on amperometric spikes as dominant-negative Cdk5 but does not affect the amperometric feet. Cells expressing Munc18 T574A have increased quantal size and broader kinetics of fusion. These results suggest that Cdk5 could, in part, control the kinetics of exocytosis through phosphorylation of Munc18, but Cdk5 also must have Munc18-independent effects that modify fusion pore conductance, which may underlie a role of Cdk5 in synaptic plasticity.     

Regulation of insulin exocytosis by calcium-dependent protein kinase C in beta cells

The control of insulin release from pancreatic beta cells helps ensure proper blood glucose level, which is critical for human health. Protein kinase C has been shown to be one key control mechanism for this process. After glucose stimulation, calcium influx into beta cells triggers exocytosis of insulin-containing dense-core granules and activates protein kinase C via calcium-dependent phospholipase C-mediated generation of diacylglycerol. Activated protein kinase C potentiates insulin release by enhancing the calcium sensitivity of exocytosis, likely by affecting two main pathways that could be linked: (1) the reorganization of the cortical actin network, and (2) the direct phosphorylation of critical exocytotic proteins such as munc18, SNAP25, and synaptotagmin. Here, we review what is currently known about the molecular mechanisms of protein kinase C action on each of these pathways and how these effects relate to the control of insulin release by exocytosis. We identify remaining challenges in the field and suggest how these challenges might be addressed to advance our understanding of the regulation of insulin release in health and disease.     

Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells

The effects of botulinum neurotoxins or their light and heavy chain subunits were investigated in digitonin-permeabilized adrenal chromaffin cells. Because these cells are permeable to proteins, the toxin had direct access to the cell interior. Botulinum type A neurotoxin and its light chain subunit inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner. The heavy chain subunit had no effect. Inhibition required introduction of the neurotoxin or light chain into the cell and was not seen when intact cells were incubated with these proteins. The inhibition of secretion by type A neurotoxin and light chain was incomplete, the maximal response being 65%. The inhibition was not overcome by increasing Ca2+ concentrations. The action of the light chain was irreversible and rapid. Botulinum type E neurotoxin also inhibited secretion in a dose-dependent manner. Its potency was increased 30-fold following mild trypsinization, which nicked the single chain protein to the dichain form. In contrast to the results seen with types A and E, botulinum type B neurotoxin did not inhibit secretion, while its light chain totally abolished secretion. Trypsinization of the neurotoxin produced the dichain form, which did not inhibit secretion. Reduction of the trypsinized neurotoxin with dithiothreitol produced inhibition equivalent to that seen with the purified light chain subunit. Isolated type A heavy chain had no effect on the inhibitory action of type A or B light chains. The data demonstrate that the ability of botulinum neurotoxins to inhibit secretion is confined to the light chain region of these proteins. Furthermore, while the botulinum neurotoxin types A, B, and E have similar macrostructures, they are not identical with respect to their biological activities.     

Quantitative investigations of amperometric spike feet suggest different controlling factors of the fusion pore in exocytosis at chromaffin cells

Around 30% of exocytosis events recorded by amperometry at carbon fiber microelectrodes exhibit a pre-spike feature (PSF) termed a “foot”. This wave is associated with the release of the neurotransmitters via a transitory fusion pore, whilst the large, main exocytotic spike is due to complete release. The amperometric data reported herein were obtained using bovine chromaffin cells stimulated with either potassium or barium ions, two commonly-employed elicitors of exocytosis. Identical trends are observed with both activators: (i) they induce the same ratio (close to 30%) of events with a foot in the population of amperometric spikes, and (ii) spikes with a foot can be divided into two primary categories, depending on the temporal variation of the current wave (viz. as a ramp, or a ramp followed by a plateau). Correlations between the characteristics of the whole current spike, and of its observed foot, have been sought; such analyses demonstrate that the maximum current of both foot and spike signals are highly correlated, but, in contrast, the integrated charges of both are poorly correlated. Moreover, the temporal duration of the PSF is fully uncorrelated with any parameter pertaining to the main current spike. On the basis of these reproducible observations, it is hypothesized that the characteristics (dimensions and topology, at least) of each secretory vesicle determine the probability of formation of the fusion pore and its maximum size, whilst molecular factors of the cell membrane control its duration, and, consequently, the amount delivered prior to the massive exocytosis of catecholamines observed as a spike in amperometry.     

Ca2+ and protein kinase C activation of mucin granule exocytosis in permeabilized SPOC1 cells

Mucin secretion by airway goblet cells is under the control ofapical P2Y₂, phospholipaseC-coupled purinergic receptors. In SPOC1 cells, the mobilization ofintracellular Ca²⁺ by ionomycin orthe activation of protein kinase C (PKC) by phorbol 12-myristate13-acetate (PMA) stimulates mucin secretion in a fully additive fashion[L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis.Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17):L201-L210, 1997]. This apparent independence between PKC andCa²⁺ in the stimulation of mucinsecretion was tested in streptolysin O-permeabilized SPOC1 cells. Thesecells were fully competent to secrete mucin whenCa²⁺ was elevated from 100 nM to3.1 µM for 2 min following permeabilization; theCa²⁺EC₅₀ was 2.29 ± 0.07 µM.Permeabilized SPOC1 cells were exposed to PMA or 4-phorbol atCa²⁺ activities ranging from 10 nMto 10 µM. PMA, but not 4-phorbol, increased mucin release at allCa²⁺ activities tested: at 10 nMCa²⁺ mucin release was 2.1-foldgreater than control and at 4.7 µM Ca²⁺ mucin release was maximal(3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 µMthan at 10 nM Ca²⁺. Hence, SPOC1cells possess Ca²⁺-insensitive,PKC-dependent, and Ca²⁺-dependentPKC-potentiated pathways for mucin granule exocytosis.

     

Comparison of protein phosphorylation patterns produced in adrenal cells by activation of cAMP-dependent protein kinase and Ca-dependent protein kinase

Bovine adrenal fasciculata cells, exposed to either ACTH or AII, synthesize glucocorticoids at an enhanced rate. It is generally accepted that the signaling pathways triggered by these two peptides are not identical. ACTH presumably acts via a cAMP-dependent protein kinase (PKA) and AII, via a calcium-dependent protein kinase. We have found that either peptide hormone stimulates synthesis of a mitochondrial phosphoprotein pp37, leading to accumulation of its proteolytically processed products pp30 and pp29. On the basis of a number of criteria, this 37 kDa protein is the bovine homolog of the 37 kDa protein that we have characterized in rodent steroidogenic tissue (Epstein L. F. and Orme-Johnson N. R.: J. Biol. Chem 266 (1991) 19,739–19,745). Further, bovine pp37 is phosphorylated when PKA or protein kinase C (PKC) is activated directly by (Bu)₂cAMP or PMA, respectively. These studies indicate that either pp37 is a common substrate for PKA and PKC in these cells or there is a common downstream kinase, which is activated by exposure to either ACTH or AII. Rat adrenal glomerulosa cells, exposed to either ACTH or AII, show an enhanced rate of mineralocorticoid synthesis. As for bovine fasciculata cells, it is thought that the signaling pathway triggered by ACTH differs from that triggered by AII. As we found for bovine fasciculata, pp37 is phosphorylated when the rat cells are exposed to either peptide hormone. However, in contrast to the finding for bovine fasciculata, while exposure of the rat glomerulosa cells to (Bu)₂cAMP does cause the synthesis of pp37, exposure of the cells to PMA does not. Taken together, these findings provide further evidence that the subcellular signaling events, triggered by the action of AII on bovine adrenal fasciculata and rat adrenal glomerulosa cells, differ. Further, the fact, that pp37 is phosphorylated only when the rate of steroidogenesis is enhanced, reaffirms its potential involvement in the signaling pathway that causes stimulation of steroid hormone biosynthesis.     

Phosphorylation of a chromaffin granule-binding protein by protein kinase C

Protein kinase C was detected in a group of Ca2+-dependent chromaffin granule membrane-binding proteins (chromobindins) on the basis of Ca2+-, phosphatidylserine-, 1,2-diolein-, and phorbol myristate acetate-stimulated histone kinase activity. When the chromobindins were incubated with [gamma-32P]ATP, Ca2+, and phosphatidylserine, 32P was incorporated predominantly into a protein of mass 37 +/- 1 kilodaltons (chromobindin 9, or CB9). Phosphorylation of this protein was also stimulated by diolein and phorbol myristate acetate, indicating that it is a substrate for the protein kinase C activity present in the chromobindins. Maximum phosphate incorporation into CB9 in the presence of 1 mM Ca2+, 75 micrograms/ml of phosphatidylserine, 2.5 micrograms/ml of diolein, and 12.5 micrograms/ml of dithiothreitol was 0.53 mol/mol of CB9 in 5 min. Eight 32P-labeled phosphopeptides were resolved in two-dimensional electrophoretic maps of trypsin digests of CB9. Phosphoamino acid analysis revealed that phosphorylation was exclusively on serine (94%) and threonine (6%) residues. Incubation of the chromobindins with chromaffin granule membranes in the presence of [gamma-32P]ATP resulted in the incorporation of 32P into eight additional proteins besides CB9 that could be separated from the membranes by centrifugation in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We suggest that phosphorylation of CB9 or these additional eight proteins may regulate events underlying exocytosis in the chromaffin cell.     

Differential effects of protein kinase C activators on carbamylcholine- and high K+-induced rises in intracellular free calcium concentration in cultured adrenal chromaffin cells

Pretreatment of adrenal chromaffin cells with protein kinase C activators, i.e. 12-O-tetradecanoyl phorbol-13-acetate (TPA) and 1-oleoyl 2-acetyl glycerol (OAG), partially inhibited carbamylcholine (CCh)-induced rise in intracellular free Ca2+ concentration ([Ca2+]i). The apparent IC50 values of TPA and OAG were 3 nM and 25 microM, respectively. The effect of TPA on the CCh-induced rise in [Ca2+]i was overcome by pretreatment of the cells with a protein kinase C inhibitor, 1-(5-isoquinidinesulfonyl)-2-methylpiperazine hydrochloride (H-7). In contrast, KCl-induced rise in [Ca2+]i was not affected by pretreating the cells with TPA or OAG. An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate failed to affect the CCh-induced rise in [Ca2+]i. CCh-induced 45Ca2+ uptake was also partially inhibited by pretreatment of the cells with TPA or OAG, but KCl-induced 45Ca2+ uptake was not affected by these pretreatments. These results indicate that protein kinase C activation causes an uncoupling of signal transduction between the nicotinic receptors and Ca2+ channels.