Chromaffin Granule Membranes Research Articles

Characterization of 14-3-3 proteins in adrenal chromaffin cells and demonstration of isoform-specific phospholipid binding.

Isoform-specific antisera were used to examine which 14-3-3 isoforms were present in bovine adrenal chromaffin cells. The eta, tau and sigma isoforms were not detectable, and the epsilon isoform was present at only low levels. 14-3-3 isoforms were readily detected with antisera against the beta, gamma and zeta isoforms. The latter isoforms were found to leak from digitonin-permeabilized chromaffin cells, as expected for cytosolic proteins, but a proportion of each isoform was retained. In subcellular fractionation studies isoforms recognized by the beta and zeta antisera were found in the cytosol and Triton-insoluble cytoskeletal fractions, while the gamma isoform was found in cytosol and also in microsomal and chromaffin granule membrane fractions. The gamma 14-3-3 protein associated with granule membranes was partially removed by a high-salt/carbonate wash, and the membranes could bind further gamma from cytosol or from a purified brain 14-3-3 protein mixture. The binding of gamma 14-3-3 was not Ca(2+)-dependent, nor was it affected by phorbol ester, GTP analogues or cyclic AMP. Using pure phospholipid vesicles it was found that gamma and also epsilon 14-3-3 proteins bound directly to phospholipids. Little binding of brain beta, eta or zeta to phospholipid vesicles was detected. Brain 14-3-3 proteins were also able to aggregate phospholipid vesicles. Recombinant 14-3-3 isoforms (tau and the Xenopus protein) were able to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized chromaffin cells. The Xenopus proteins lacks part of the extreme N-terminus, indicating that this domain is not essential for function in exocytosis.

Monoclonal antibodies reactive with a monoamine transporter preparation purified from bovine adrenal chromaffin granule membranes

There have been no reports of monoclonal antibodies reactive with vesicular monoamine transporters from any source. Western blotting and ELISA data obtained using polyclonal serum from a mouse immunized with a highly purified bovine chromaffin granule monoamine transporter preparation yielded data consistent with the presence of antibodies to the transporter. Hybridomas produced by polyethylene glycol fusion of spleen cells from the mouse with X63/Ag8.653 myeloma cells were screened in an ELISA using partially purified transporter as coating agent. Of the 1142 wells containing colonies, 14 were positive in the initial screen. Hybridomas from wells testing positive were transferred to 24-well plates, grown up, and rescreened. Those still testing positive were subcloned, and the resulting positive wells containing single colonies were grown up and stocked. Of the 6 positive clones that tolerated freeze/thaw (0.5% of the wells tested), 1 was IgGI k , 2 were IgG2a k , and 3 were IgG2b k isotypes. Ascites fluid was generated in pristane-primed BALB/c mice using hybridomas that had been cloned 2–3 times, and antibodies purified on immobilized Protein A. Immunoreactivity with a mixture of these antibodies, or with only one of them, coincided with dihydrotetrabenazine (TBZOH) binding activity in fractions eluted from all columns employed in the transporter purification. Antibody from at least one of the clones was capable of removing [ 3H]TBZOH binding activity from a partially purified preparation of transporter. Monoclonal antibodies exhibiting these properties have not been reported previously.

Bovine chromaffin cells: Studies on the biosynthesis of phospholipids in chromaffin granules

We have studied the biosynthesis of chromaffin granules by labelling primary cultures of bovine chromaffin cells with either [ 35S]methionine or various precursors for lipids. After labelling the cells were subjected to subcellular fractionation including density gradient centrifugation. After [ 35S]methionine significant label (mainly represented by labelled chromogranin A) was found in the soluble proteins of chromaffin granules, whereas the membranes were relatively little labelled. However incorporation into membrane bound dopamine β-hydroxylase and cytochrome b-561 could be demonstrated. Neither of the used lipid precursors ([ 3H]glycerol, [ 3H]choline, [ 3H]palmitic acid or [ 3H]arachidonic acid) was incorporated to any significant extent into the soluble components of chromaffin granules. Thus there is no evidence that this secretory compartment contains any lipids or acylated proteins. Incorporation of lipid precursors into the membranes of chromaffin granules was apparently low. After short chases labelled lysolecithin was not present in these organelles. However with prolonged chase times labelled lysolecithin apparently appeared in chromaffin granules irrespective of whether the cells were stimulated or not. We can conclude that the reusable membranes of chromaffin granules have a very low lipid turnover. Lysolecithin is not transferred into these organelles during biosynthesis but is formed in them during their long life span. This formation of lysolecithin is independent of stimulation of these cells and therefore unlikely to be involved in exocytosis.

Disaccharide Composition of Heparan Sulfates: Brain, Nervous Tissue Storage Organelles, Kidney, and Lung

We have characterized the structural properties of heparan sulfates from brain and other tissues after depolymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum. The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards. In rat, rabbit, and bovine brain, 46-69% of the heparan sulfate disaccharides are N-acetylated and unsulfated, and 17-21% contain a single sulfate residue in the form of a sulfoamino group. In rabbit, bovine, and 1-day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N-sulfate account for an additional 10-14%, together with smaller and approximately equal proportions (5-9%) of mono-, di-, and trisulfated disaccharides having sulfate at the 6-position of the glucosamine residue. Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6-sulfated N-acetylglucosamine residues. In chromaffin granules, the catecholamine- and peptide-storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5-10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71%) or monosulfated (18%). In sympathetic nerves, norepinephrine is stored in large dense cored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules. However, in contrast to chromaffin granules, heparan sulfate accounts for approximately 75% of the total glycosaminoglycans in large dense-cored vesicles and more closely resembles heparin, insofar as it contains only 21% unsulfated disaccharides, 10% mono- and disulfated disaccharides, and 69% trisulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of Laminin B Chain-Like Protein in Bovine and Rat Adrenal Chromaffin Granules

The presence of a glycoprotein laminin in bovine adrenal chromaffin granules was examined by SDS-PAGE followed by immunoblotting. The two chromaffin granule membrane fractions were obtained by linear sucrose gradient centrifugation followed by freezing and thawing and gel-filtration of the chromaffin granule-rich fraction, respectively. The purity of the granules in these fractions was examined by electron microscopy. These fractions contained laminin B chain-like immunoreactivity as a major immunoreactive component against anti-laminin. Laminin A chain-like immunoreactive protein was undetectable. The soluble fraction of the chromaffin granules contained no immunoreactive peptide. The presence of laminin-like immunoreactivity in the chromaffin granules was confirmed by immunocytochemical study. Laminin B chain-like immunoreactivity was also identified in the rat adrenal chromaffin granule fraction. Laminin A chain was hardly detected, as in the case of bovine adrenals. Structure of laminin in chromaffin granules in bovine and rat adrenals may be different from that of mouse Englebrethe-Holm-Swarm sarcoma laminin. The functional significance of laminin B chain-like protein in the granules is unknown at present.

Role of Mg-ATP in norepinephrine biosynthesis in intact chromaffin granules.

Dopamine beta-monooxygenase converts dopamine to norepinephrine in intact chromaffin granules using intragranular ascorbic acid as a cosubstrate. Mg-ATP with external ascorbic acid is required for maximal norepinephrine biosynthesis. Mechanisms to explain these requirements were investigated specifically using intact granules. The effect of Mg-ATP was independent of membrane potential (delta psi) because norepinephrine biosynthesis was unchanged whether delta psi was positive or collapsed. Furthermore, the effect of Mg-ATP was independent of absolute intragranular and extragranular pH as well as the pH difference across the chromaffin granule membrane (delta pH). Nevertheless, norepinephrine biosynthesis was inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzofurazane, and N,N-dicyclohexylcarbodiimide, specific inhibitors of the secretory vesicle ATPase that may directly affect proton pumping. Biosynthesis occurred normally with other ATPase inhibitors that do not inhibit the ATPase in secretory vesicles. The data indicate that the effect of Mg-ATP with ascorbic acid is mediated by the granule membrane ATPase but independent of maintaining delta psi and delta pH. An explanation of these findings is that Mg-ATP, via the granule ATPase, may change the rate at which protons or dopamine are made available to dopamine beta-monooxygenase.

Uptake of the Neurotoxin, 4-MethylphenylPyridinium, into Chromaffin Granules and Synaptic Vesicles: A Proton Gradient Drives Its Uptake through Monoamine Transporter

Energy dependence for uptake of 4-methyphenyl-pyridinium (MPP +), a neurotoxin causing Parkinsonism-like symptoms, by adrenal chromaffin granule membrane vesicles and brain synaptic vesicles was studied. The compound was actively taken up by the chromaffin vesicles dependent on hydrolysis of ATP with a K m value of 22 μM and maximum velocity of 2.9 nmol/min/mg protein. The uptake was sensitive to reserpine (1 μM) and bafilomycin (50 nM) (inhibitors of the vesicular monoamine transporter and vacuolar-type H +-ATPase, respectively) and substrates for monoamine transporters, but insensitive to imipramine (an inhibitor of the monoamine transporter present in the plasma membrane). The uptake was greatly reduced upon dissipation of the proton gradient by ammonium ion or nigericin with KCl, but stimulated 1.6-fold by valinomycin plus K +. Dissipation of the proton gradient also induced rapid efflux of MPP + from the vesicles. The MPP + (monoamine) transporter was solubilized from chromaffin vesicles and reconstituted into liposomes with purified bacterial F 0F 1-ATPase. MPP + was taken up by the liposomes coupled with ATP hydrolysis by F 0F 1, and the uptake was sensitive to reserpine, dissipation of the proton gradient, and azide. Brain synaptic vesicles also accumulated MPP +, showing similar kinetics, inhibitor sensitivities, and energy coupling to those of chromaffin vesicles. Furthermore, MPP + inhibited the uptake of dopamine without affecting the uptake of glutamate or γ-aminobutyrate. These results indicated that MPP + was taken up through the reserpine-sensitive monoamine transporter into chromaffin vesicles and synaptic vesicles and that the energy for accumulation of MPP + was supplied as a proton gradient (acidic inside) established by H +-ATPase.

Exocytosis in chromaffin cells. Possible involvement of the heterotrimeric GTP-binding protein G(o)

The use of non-hydrolyzable analogues of GTP in permeabilized secretory cells suggests that guanine nucleotide-binding regulatory proteins (G proteins) may be involved in regulated exocytosis. Because GTP analogues are known to modulate both monomeric low molecular mass G proteins and heterotrimeric G proteins, we have examined the effect of mastoparan, an activator of heterotrimeric G proteins, on secretion from intact and permeabilized chromaffin cells. In intact cells, mastoparan inhibited catecholamine secretion evoked by nicotine but had no effect on release induced by other secretagogues. In permeabilized cells, mastoparan inhibited calcium-dependent secretion providing that the pores created in the plasma membrane allow the penetration of the peptide into the cytoplasm. These results indicate that mastoparan blocks the exocytotic machinery through an intracellular target protein that may not be located just beneath the plasma membrane. Accordingly, mastoparan was able to stimulate G proteins associated with purified chromaffin granule membranes, in a range of concentration and Mg2+ requirement that was similar to its inhibitory effect on secretion. Mas 17, a mastoparan analogue inactive on purified G proteins, neither modified catecholamine secretion nor stimulated chromaffin granule G proteins. The substance P-related peptide, GPAnt-2, known to antagonize the effects of mastoparan on G(o), blocked both the inhibitory effect of mastoparan on secretion and the mastoparan-stimulated GTPase activity in chromaffin granule membranes. Moreover, specific antibodies raised against the carboxyl terminus of G(o) alpha reversed in a dose-dependent manner the inhibition by mastoparan on catecholamine release and the stimulation by mastoparan of chromaffin granule-associated G proteins. These results suggest that the secretory machinery in chromaffin cells can be blocked by activating a G(o) protein. Consistent with this finding, two other known activators of heterotrimeric G proteins, aluminum fluoride and benzalkonium chloride, inhibited calcium-evoked catecholamine secretion in streptolysin O-permeabilized chromaffin cells. We conclude that an inhibitory G(o) protein, possibly located on the membrane of secretory granules, is involved in the final stages of exocytosis in chromaffin cells.

Characterization of glycoprotein II from bovine adrenal medullary chromaffin granules. Identification of components representing the secretory vesicle counterparts of the lysosomal-associated membrane glycoproteins (lamp-1 and lamp-2)

Glycoprotein II (GpII) is a heterogenous glycoprotein isolated from the membranes of bovine chromaffin granules in the adrenal medulla. When viewed by two-dimensional electrophoresis this glycoprotein consists of two components, upper (GpIIa) and lower (GpIIb), with a molecular mass of 80,000-100,000 daltons and a pI of 4.2-4.7. NH2-terminal sequence analysis of GpIIa and GpIIb revealed sequence similarity with lysosomal membrane glycoproteins (lamp-1 and lamp-2), which was supported by sequence data of peptides from trypsin and cyanogen bromide digestions. An oligonucleotide probe was used to isolate a cDNA clone encoding the nucleotide sequence of GpIIa. The predicted amino acid sequence of GpIIa shares a 72% identity with the human lamp-1 type protein, which belongs to a highly conserved group of lysosomal-associated membrane glycoproteins (lamp proteins), whose function is still unknown. The COOH-terminal region of GpIIa was identical to the COOH-terminal region of lamp proteins. This COOH-terminal determinant has been demonstrated to be essential for the intracellular targeting of lamp proteins to lysosomes. A synthetic peptide antisera to the COOH-terminal region of GpIIa was used to show that this region is present on purified chromaffin granules and not proteolytically processed. The sequence analysis of GpIIa and immunological data confirm GpII as the secretory granule counterpart of lamp proteins and raise some questions regarding intracellular targeting between lysosomes and secretory granules within the chromaffin cell.

P-442 - Ionic factors affecting the interaction between soluble tyrosine hydroxylase and chromaffin granule membranes.

P-442 - Ionic factors affecting the interaction between soluble tyrosine hydroxylase and chromaffin granule membranes.

Modification of arginyl or histidyl groups affects the energy coupling of the amine transporter.

We have characterized the effects of phenylglyoxal and diethyl pyrocarbonate (DEPC) on the catalytic cycle of the amine transporter in chromaffin granule membrane vesicles. Both reagents inhibited transport in a dose-dependent reaction (with IC50 values of 8 and 1 mM, respectively). The inhibition by DEPC was specific for histidyl groups since transport could be restored by treatment with hydroxylamine. Neither phenylglyoxal nor DEPC inhibited binding of either R1- or R2-type ligands, indicating that the inhibition of transport is not due to a direct interaction with either of the known binding sites. Interestingly, however, the acceleration of reserpine binding (an R1 ligand) by a transmembrane H+ gradient is inhibited by both reagents at concentrations identical to those which inhibit transprot. As previously demonstrated, transport of one proton across the transporter is required for this acceleration to take place [Rudnick, G., Steiner-Mordoch, S., Fishkes, H., Stern-Bach, Y., & Schuldiner, S. (1990) Biochemistry 29, 603-608]. Therefore, we suggest that either proton transport or a conformational change induced by proton transport is inhibited by both types of reagents.

Regulation of the chromaffin granule catecholamine transporter in cultured bovine adrenal medullary cells: stimulus-biosynthesis coupling.

The transsynaptic induction of the monoamine transporter present on the membrane of chromaffin granules was studied in primary cultures of dissociated bovine adrenomedullary cells submitted to a chronic secretory stimulation. The amount of the vesicular monoamine transporter was assayed by binding of the specific ligand [3H]-dihydrotetrabenazine. After several days of incubation in the presence of high potassium, the concentration of [3H]-dihydrotetrabenazine binding sites was increased by a 1.5-2.5 factor. This increase was smaller in the presence of the cholinergic agonist carbachol. The long-term inductions of the vesicular monoamine transporter, of tyrosine hydroxylase, and of acetylcholinesterase were of similar magnitude. Under the same conditions, we found no variation in either the activities of other catecholamine biosynthetic enzymes (dopamine beta-hydroxylase and DOPA decarboxylase), or in metabolic enzymes such as lactate dehydrogenase and cytochrome c oxidase, and a decrease in the cellular content of chromogranin A and cytochrome b-561. The induction of the vesicular monoamine transporter was inhibited by the calcium channel antagonists, fluspirilene and nifedipine, and was increased by the agonist Bay K 8644. It was abolished by cycloheximide and actinomycin D. These results indicate that calcium entry into chromaffin cells increases the synthesis of the vesicular monoamine transporter, presumably by transcriptional activation. Elevation of intracellular cyclic AMP concentration or activation of protein kinase C also induced an increase in the expression of the vesicular monoamine transporter. Our results confirm that components of storage vesicle membranes are differentially regulated in response to secretory stimulation, as are several cytosolic or intravesicular soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-independent K(+)-selective channel from chromaffin granule membranes.

Intact adrenal chromaffin granules and purified granule membrane ghosts were allowed to fuse with acidic phospholipid planar bilayer membranes in the presence of Ca2+ (1 mM). From both preparations, we were able to detect a large conductance potassium channel (ca. 160 pS in symmetrical 400 mM K+), which was highly selective for K+ over Na+ (PK/PNa = 11) as estimated from the reversal potential of the channel current. Channel activity was unaffected by charybdotoxin, a blocker of the [Ca2+]-activated K+ channel of large conductance. Furthermore, this channel proved quite different from the previously described channels from other types of secretory vesicle preparations, not only in its selectivity and conductance, but also in its insensitivity to both calcium and potential across the bilayer. We conclude that the chromaffin granule membrane contains a K(+)-selective channel with large conductance. We suggest that the role of this channel may include ion movement during granule assembly or recycling, and do not rule out events leading to exocytosis.

Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules.

An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla. These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones. A number of experiments indicated that these glycoproteins were associated. These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules. Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins. Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes. Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114. It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane. This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules.

Interaction of dopamine-β-monooxygenase with chromaffin granule membrane lipids

The interaction between bovine adrenal medullary dopamine-β-monooxygenase and liposomes from chromaffin granule membrane lipids as a function of pH, lipid and salt concentration was studied by ultracentrifugation. Efficient adsorption of dopamine-β-monooxygenase to liposomes occurs in the pH range 5.0–6.5 and at low ionic strength. The adsorption was not detected in the case of apoenzyme. The membrane dopamine-β-monooxygenase forms a complex with liposomes more effective than soluble does. The data obtained lead to certain conclusions about the specificity of complex between the enzyme and liposomes.

EM localization of chromaffin cell ATPase

During exocytosis of the chromaffin granules, ATP is released. ATP can then be hydrolyzed by the ecto-ATPases of the plasma membrane to provide adenosine for reuptake or for activation of P1purinoceptors. Chromaffin granule membranes also possess ATPase activity. This activity is linked to the uptake of catecholamines from the cytoplasm into the membrane-bound granule compartment.In this report we combine EM cytochemistry and immunogold labelling to provide further evidence for the presence of ATPase on both the plasma membrane and granule membranes of bovine chromaffin cells in culture.

Membrane recapture and early triggered secretion from the newly formed endocytotic compartment in bovine chromaffin cells.

1. Recycling of secretory vesicles in cultured bovine adrenal medullary cells was investigated. 2. Extracellular horseradish peroxidase (HRP), a fluid phase marker, was taken up into cultured adrenal medullary cells following carbamylcholine-induced secretion of catecholamine. 3. The endocytosed HRP remained compartmentalized within the cell, migrating to a low density band on a Percoll density gradient. The endocytotic compartment was distinct from the major pool of catecholamine-containing chromaffin granules, which were found at much higher densities on the Percoll gradient. 4. The chromaffin granule membrane marker dopamine beta-hydroxylase was associated with the endocytosed HRP compartment as well as with the heavier chromaffin granules. 5. A subsequent challenge of the cells with carbamylcholine triggered the release of up to forty per cent of the endocytosed HRP. 6. The time course for secretion of the fluid phase marker was similar to that for catecholamine secretion. 7. Triggered release of HRP was dependent on extracellular calcium. The dependence on the extracellular calcium concentration was similar to that of catecholamine release. 8. Release of HRP could be triggered from electropermeabilized cells by raising the intracellular Ca2+ into the micromolar range. The intracellular Ca2+ dependence of triggered HRP release was similar to that for catecholamine release. 9. HRP could be secreted as early as 5 min, and as late as 2 h after endocytosis. 10. These data provide evidence that endocytotic vesicles can rapidly re-enter the secretory cycle. Endocytosed vesicles may therefore not have to recycle via the trans-Golgi reticulum to form high-density chromaffin granules in order to re-enter the regulated secretory pathway.

P-Chloroamphetamine induces serotonin release through serotonin transporters.

p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.

Organization of cortical cytoskeleton of cultured chromaffin cells and involvement in secretion as revealed by quick-freeze, deep-etching, and double-label immunoelectron microscopy

We have studied the organization of the cytoskeleton in both unstimulated and stimulated cultured chromaffin cells, as well as its relationship with their secretory process by exocytosis. We found the spatial heterogeneity in the intensity of cortical rhodamine-phalloidin staining within a cell. The overall staining pattern or intensity was minimally altered after stimulation, although dopamine-beta-hydroxylase (DBH) antigen, a marker for the chromaffin granule membrane, was exposed preferentially on the plasma membrane areas with lower intensity of rhodamine-phalloidin staining. Using the quick-freeze, deep-etch technique, we found the heterogeneity in the organization of cortical cytoskeletal networks--some regions have actin filament bundles running parallel to the plasma membrane interspersed between granules and the plasma membrane, while others have few actin filaments beneath the plasma membrane before stimulation. Actin filaments were rarely observed in the inner cytoplasm. We did not observe the overall change in its organization after stimulation. Double-label immunogold EM using anti-DBH antibody and anti-actin antibody combined with statistical analysis showed that (1) DBH was exposed on the plasma membrane preferentially where actin was sparse after stimulation (significant at less than 0.1%), although (2) regions having sparse actin were not always the sites for DBH exposure, and (3) the cortical actin zone was sometimes disrupted at the DBH-exposed sites after stimulation. The present data suggested that (1) secretion is related to heterogeneous organization of cortical cytoskeleton after stimulation and (2) massive synchronized reorganization of the cytoskeleton in the whole cell is not necessary for secretion, although small changes of the cytoskeleton might occur under local regulation at each exocytotic site at the moment of the release.

Kex2-like proteolytic activity in adrenal medullary chromaffin granules

This study demonstrates the presence of boc-Gln-Arg-Arg-MCA cleaving activity in bovine chromaffin granule membranes that resembles yeast Kex2 proteolytic activity. The chromaffin granule boc-Gln-Arg-Arg-MCA cleaving activity, like Kex2 proteolytic activity, shows calcium dependence, optimum activity at pH 7.5-8.2, inhibition by serine protease inhibitors, and preference for cleavage at the COOH-terminal side of Arg-Arg and Lys-Arg, over Lys-Lys, paired basic residues. Potent inhibition by the active-site directed inhibitor [D-Tyr]-Glu-Phe-Lys-Arg-CK (20 microM) provided further evidence for dibasic residue cleavage site specificity. These results are the first report of endogenous mammalian Kex2-like proteolytic activity that may be related to PC1/PC3 and PC2 enzymes, the newly discovered mammalian homologues of Kex2 protease. It will be important to determine the role of this Kex2-like proteolytic activity in processing the precursors of adrenal medullary neuropeptides.