The Annexin II-p11 Complex Is Involved in Regulated Exocytosis in Bovine Pulmonary Artery Endothelial Cells*

Annexin II is a member of a multigene family of Ca2+-regulated, membrane-binding proteins implicated through biochemical and perforated cell experiments in Ca2+-triggered secretion. Within most cells annexin II resides in a tight heterotetrameric complex with a cellular protein ligand, p11, and complex formation is mediated via the N-terminal 14 residues of annexin II including the N-terminal acetyl group. To analyze at the single cell level whether the annexin II-p11 complex is involved in regulated secretion, we used membrane capacitance measurements to follow exocytotic fusion events in bovine aortic endothelial cells manipulated with respect to their annexin II-p11 complex formation. Upon guanosine 5′-O-(thiotriphosphate) (GTPγS) stimulation, the endothelial cells show a significant increase in membrane capacitance which is generally preceded by a transient rise in intracellular Ca2+ and thus indicative of the occurrence of Ca2+-regulated secretion. The GTPγS-induced capacitance increase is markedly reduced in cells loaded with a synthetic peptide, Ac1–14, which corresponds in sequence to the N-terminal 14 residues of annexin II in their correctly acetylated form and which is capable of disrupting preformed annexin II-p11 complexes. The effect of the peptide is highly specific as the nonacetylated variant, N1–14, which is incapable of disrupting annexin II-p11, does not interfere with the GTPγS-induced increase in membrane capacitance. These data show that intact annexin II-p11 complexes are indispensable for regulated exocytosis to occur in an efficient manner in endothelial cells.

Changes in the intracellular Ca 2ϩ levels are known to regulate a variety of cellular processes ranging from the control of cell architecture to intracellular communication, cellular differentiation, and cell death. These cellular responses are mediated through a large number of Ca 2ϩ -binding proteins which are thought to couple the Ca 2ϩ stimulus (transient increases in the cytosolic Ca 2ϩ concentration) to the different biological events most likely by displaying different properties in their Ca 2ϩ -free and their Ca 2ϩ -bound conformations. Generally, the Ca 2ϩ -binding proteins are grouped according to the structure of their principal Ca 2ϩ binding motif with the two largest families known to date being the EF hand proteins and the annexins (1). In contrast to EF hand-type and most other Ca 2ϩ -binding pro-teins, annexins characteristically also display a Ca 2ϩ -dependent interaction with negatively charged phospholipids enriched in the cytosolic leaflets of cellular membranes (2)(3)(4). Due to these biochemical characteristics, annexins have been implicated in mediating and/or regulating membrane related events including membrane-membrane contacts, membrane-cytoskeleton linkages, membrane organizations, and ion currents across membranes. However, experiments addressing the problem of annexin function in living cells have been very scarce, and the biological role of annexins remains enigmatic.
Annexin II is a 36-kDa protein comprising two principal domains, a C-terminal protein core consisting of four homologous segments of 70 amino acids, the so-called annexin repeats, and an N-terminal region which is less structured and 30 residues in length (4). While the core domain harbors binding sites for Ca 2ϩ , phospholipid (the common annexin ligands) and F-actin, the N-terminal sequence contains phosphorylation sites for pp60 src and protein kinase C as well as the binding site for an intracellular protein ligand of 11 kDa. This p11 protein is a member of the S100 family of EF hand-type Ca 2ϩ -binding proteins which forms obligatory dimers (1,5). p11 binding is mediated solely through the N-terminal 14-amino acids of annexin II which form an amphiphatic ␣-helix with the hydrophobic side chains and the N-terminal acetyl group of the posttranslationally modified annexin II being indispensable for high affinity p11 interaction (6,7). Complex formation establishes a heterotetrameric annexin II 2 p11 2 entity, which has altered biochemical properties when compared with monomeric annexin II, e.g. the complex but not monomeric annexin II can aggregate chromaffin granules at low micromolar Ca 2ϩ concentrations (8).
Within cells the annexin II-p11 complex is found in the cortical cytoskeleton underlying the plasma membrane, on early endosomal membranes as well as associated with secretory vesicles like chromaffin granules (4). Monomeric annexin II, on the other hand, appears to be at least partly cytosolic. Its complex formation with p11 and thus the cell cortex association is regulated by protein kinase C phosphorylation in the Nterminal domain (9 -11). In line with the intracellular distribution, annexin II-p11 has been implicated in early endocytotic and late secretory events occurring at or close to the plasma membrane (4). A role in Ca 2ϩ -regulated exocytosis has been deduced mainly from studies using digitonin-permeabilized chromaffin cells. Here the progressive loss of secretory responsiveness to elevated Ca 2ϩ concentrations can be slowed down by the addition of annexin II with the annexin II-p11 complex being more efficient than monomeric annexin II (12,13). The suggested role of annexin II in Ca 2ϩ -regulated exocytosis is in line with the morphological observation that in quick-frozen deep-etched chromaffin cells the protein appears to form physical connections between the plasma membrane and the membrane of secretory granules (14). However, annexin II-p11 is probably not an obligatory and general constituent of Ca 2ϩ regulated secretory pathways since the protein complex shows variable expression levels in different cell types with exocytotically active neuronal cells containing little annexin II and since ectopic expression of a trans-dominant annexin II-p11 mutant in rat pheochromocytoma PC12 cells does not affect the Ca 2ϩtriggered dopamine release in a permeabilized cell system (15).
To analyze within living cells expressing considerable amounts of annexin II-p11 whether the complex participates in regulated exocytosis we chose bovine pulmonary artery endothelial cells as a model system and used membrane capacitance tracings to follow exocytotic fusion events. We show that the cells, which contain as typical secretory granules Weibel-Palade bodies positive for von Willebrand factor, respond to a GTP␥S 1 stimulus by a transient rise in cytosolic Ca 2ϩ and a subsequent increase in cell surface area by 15%. Loading of the cells with a synthetic peptide comprising the N-terminal 14 residues of annexin II including the N-acetyl group (Ac1- 14) significantly interferes with the GTP␥S-induced membrane capacitance increase. This peptide harbors the entire p11 binding site and is capable of disrupting preformed annexin II-p11 complexes. In contrast to Ac1-14, the nonacetylated 14-mer (N1-14) does not interfere with annexin II-p11 complex formation and also has no effect on the GTP␥S induced capacitance increase. The specific effect of the complex disrupting annexin II peptide indicates that annexin II-p11 participates in regulated exocytosis in endothelial cells possibly by providing physical linkages at the level of the plasma membrane.

EXPERIMENTAL PROCEDURES
Antibodies and Proteins-Mouse monoclonal antibodies, H28 and H21, were used for the detection of annexin II and p11, respectively (16). Von Willebrand factor was stained with a polyclonal rabbit antibody (Dako). Dichlorotriazinyl aminofluorescein-coupled goat antimouse, rhodamine-coupled goat anti-rabbit, and Texas Red-coupled goat anti-rabbit antibodies (Dianova) were employed as secondary antibodies. All secondary antibodies were tested in control experiments by omitting the primary antibody and showed neglectable fluorescence signals on CPAE cells. Immunoblotting experiments employed horseradish peroxidase-coupled pig anti-mouse antibodies (Dako) as secondary antibodies.
The heterotetrameric annexin II-p11 complex was purified from porcine intestinal epithelium (17). Monomeric annexin II was expressed recombinantly in baculovirus-infected insect cells and purified from the cell lysate as described previously (18).
Peptides-Peptide synthesis was carried out on an automated synthesizer (model 9050; Milligen). For N-terminal acetylation, peptides were left on the resin, and the Fmoc (N-(9-fluorenyl)methoxycarbonyl) group was released by treatment with dimethylformamide. Subsequently, the free N terminus was acetylated with acetic anhydride/Nethyldiisopropylamine for 30 min. Removal of the protection group was then achieved by treatment with 95% trifluoroacetic acid, 2.5% ethanediol, 2.5% anisole. Peptides were purified by reverse-phase high performance liquid chromatography on a preparative column (Vydac 218TP 1022) using a linear acetonitrile gradient for elution. All products were characterized by mass spectrometry (MALDI compact III; Kratos Analytical) and by N-terminal sequence analysis of a nonacetylated aliquot on an automated gas-phase sequenator (Knaur model 810). The purified peptides were lyophilized, washed three times with water, and then stored at Ϫ20°C as lyophilized aliquots.
Cell Culture-Experiments were performed on single, nonconfluent endothelial cells of an established cell line from bovine pulmonary artery (cell line CPAE, ATCC CCL-209). Cells were grown in Dulbecco's modified Eagle's medium containing 20% fetal calf serum, 2 mM glutamine, 100 g/ml streptomycin, 100 units/ml penicillin, detached by exposure to 0.05% trypsin in a Ca 2ϩ -and Mg 2ϩ -free solution, reseeded on gelatin-coated coverslips, and kept in culture 2-4 days before use.
Immunofluorescence-CPAE cells grown on fibronectin-coated glass coverslips were washed briefly in phosphate-buffered saline and fixed and permeabilized for 10 min in methanol at Ϫ20°C. Subsequently, the cells were washed three times, 5 min each, in phosphate-buffered saline and incubated in a moist chamber with 20 l of the primary antibody for 30 min at 37°C. The monoclonal antibodies H28 and H21 were employed as undiluted hybridoma culture supernatants, whereas the polyclonal anti-von Willebrand factor antibody was diluted 1:100. After washing in phosphate-buffered saline (three times, 5 min each), the cells were incubated with the respective secondary antibodies, washed again, and mounted in Moviol. Conventional microscopy was performed using a Zeiss Axiophot photomicroscope and Kodak Tmax 3200 film. Confocal images were taken using a Leica laser scanning microscope.
Capacitance and Intracellular Ca 2ϩ Measurements-Cells were voltage clamped in the whole-cell patch clamp mode by use of an EPC-9 patch clamp amplifier (Heka, Germany) technique. Holding potential was 0 mV. The normal bath solution contained (in mM): 150 NaCl, 6 KCl, 1 MgCl 2 , 1.5 CaCl 2 , 10 glucose, 11.5 HEPES, pH adjusted to 7.4 with NaOH. The osmolarity of the solution, as measured with a vapor pressure osmometer (Wescor 5500, Schlag, Gladbach, Germany), was 315 mOsm. The pipette solution contained (in mM): 40 KCl, 100 potassium aspartate, 1 MgCl 2 , 0.5 EGTA, 4 Na 2 ATP, 10 HEPES, pH adjusted to 7.2 with KOH. Under these conditions, no K ϩ currents can be activated. For stimulation of exocytosis, cells were loaded after breaking the membrane seal with 100 M GTP␥S.
For capacitance measurements, we used a double sine wave method to determine the electrical capacitance of the cell surface. The system was based on a TMS320C30 (Texas Instruments) floating point Digital Signal Processor board. Two sine waves of 125 and 500 Hz and 5 mV amplitude were alternatively imposed on the membrane. Current and voltage were sampled at a frequency of 17,280 Hz via a multiplexer circuit, which allows the use of identical circuits for amplifying and filtering of both signals and minimizes the instrumental phase shifts. The length of the time series was 864 data points, and consisted of 12 and 18 sine wave periods for each frequency. Membrane capacitance calculations were based on a lumped circuit composed of a parallel arrangement of the equivalent membrane capacitance (C m ) and resistance (R m ) in series with the access resistance R s . The use of two sine waves enables the calculation of these three parameters with standard AC analysis formalism. The present version of the method allows for updating these parameters at a rate of three estimations per s. The stray capacitance of the system, as determined with a dummy resistive network, was less than 0.8 pF. For the electrodes used in these experiments (between 2 and 5 megohms) we measured access resistances between 6 and 18 megohms. These values were rather stable during cell swelling. Using a test circuit, we obtained by changing the access resistance between 3 and 35 megohms only a deviation of the measured capacitance from the circuit capacitance between 1 and 1.6%.
For [Ca 2ϩ ] i measurements cells on the coverslips were loaded with 2 M fura-2/AM and then washed three times in the experimental chamber with Krebs solution to remove extracellular fura-2/AM. Changes in intracellular Ca 2ϩ were monitored by means of a photo multiplier-based system, consisting of an inverted microscope (Zeiss IM 10), filter wheel, amplifier and controller (Luigs & Neumann, Germany), and a photo multiplier unit (Hamamatsu, Japan). Cells were illuminated alternatively at excitation wave lengths of 360 and 390 nm through a rotating filter wheel (speed between 2 and 3 revolutions/s). Auto fluorescence was measured on cell-free parts of the coverslips and was automatically subtracted from the Ca 2ϩ signals. The apparent concentration of free Ca 2ϩ was calculated from the fluorescence ratio R i (360/390 nm): where K eff is the "effective binding constant," R 0 the fluorescence ratio at "low" Ca 2ϩ (1 mM EGTA) and R 1 that at high Ca 2ϩ (1 mM). Phospholipid Binding-Phospholipid vesicles were prepared from bovine brain extract as described previously (19). Binding studies employing monomeric annexin II or the annexin II-p11 complex in the presence or absence of the peptides Ac1-14 and N1-14 were carried out using 0.25 mg/ml liposomes in 100 l of a buffer containing 50 mM imidazole/HCl, pH 7.5, 150 mM NaCl, and EGTA-buffered Ca 2ϩ to give a final free Ca 2ϩ concentration of 250 nM. In experiments analyzing the petide effects, 5.5 pmol of annexin II-p11 were incubated with a 500-fold molar excess of the respective peptide in assay buffer for 5 min at room temperature prior to adding the liposomes. Subsequently, the mixture was incubated for 25 min at room temperature and liposome-bound protein was separated from the unbound fraction by centrifugation at 150,000 ϫ g for 15 min. The resulting phospholipid pellet was resus-pended in 20 l of SDS sample buffer (20), whereas the supernatants obtained were dried down and then dissolved in 20 l of SDS sample buffer. 10 l of each sample were analyzed by SDS-polyacrylamide gel electrophoresis (20) and immunoblotting (21).

GTP␥S Induced Secretion in Cultivated Endothelial Cells-
Calf pulmonary artery endothelial (CPAE) cells were chosen to analyze the participation of annexin II-p11 in regulated exocytosis for two major reasons. 1) Within the mammalian organism cells of the endothelium show the strongest expression of annexin II and we have shown previously that CPAE cells contain significant amounts of annexin II and p11 (22). 2) Cultivated endothelial cells are capable of responding to an external stimulus, e.g. thrombin, by Ca 2ϩ -triggered exocytosis of Weibel-Palade bodies, the principal storage granules of von Willebrand factor (vWF) and P-selectin (23). Fig. 1 shows that the CPAE cells used in this study contain vWF-positive granules with the typical morphological appearance of Weibel-Palade bodies. Moreover, treatment with the Ca 2ϩ ionophore A23187 induces cell surface appearance of vWF as revealed by immunofluorescence staining of nonpermeabilized cells (not shown). This indicates that the cells are capable of Ca 2ϩ -regulated exocytosis of at least vWF-positive granules.
To document exocytosis at the single cell level we employed membrane capacitance analyses. Cells were challenged with GTP␥S, which was shown previously to induce intracellular Ca 2ϩ rises and to effectively trigger Ca 2ϩ regulated exocytosis in a number of different cells including Chinese hamster ovary, mast, and chromaffin cells as well as pancreatic ␤ cells (24 -27). Fig. 2 reveals that the infusion of GTP␥S (100 M) produces an increase in endothelial cell capacitance. Simultaneous measurement of the intracellular free Ca 2ϩ concentration, [Ca 2ϩ ] i , shows that the rise in membrane capacitance is generally preceded by a Ca 2ϩ transient of approximately 250 nM as compared with the Ca 2ϩ concentration of less than 100 nM in resting cells. This GTP␥S-induced Ca 2ϩ mobilization is observed in most but not all cells. A fraction of approximately 15% of the endothelial cells analyzed responds to the GTP␥S stimulus by capacitance increase without an apparent change in the intracellular Ca 2ϩ levels (not shown). However, in all experiments a holding potential of 0 mV was chosen, and this may reduce Ca 2ϩ influx because of a reduced driving force. Nevertheless, our data are in line with previous studies in other cell systems, indicating that GTP␥S can also trigger exocytosis without liberation of Ca 2ϩ from intracellular stores (26, 28 -30).
Our data show that the surface area of CPAE cells is increased by 15-20% upon GTP␥S stimulus. Given the scale of this significant membrane increase it is unlikely to have resulted from a block of endocytotic membrane uptake occurring on a background of ongoing constitutive exocytosis. Moreover, constitutive exocytosis is inhibited by GTP␥S in other cells (31), arguing against a massive increase in surface area by constitutive membrane flow, even if endocytosis were inhibited under the conditions chosen. Thus, the significant increase in plasma membrane capacitance observed in GTP␥S treated endothelial cells most likely results from an induction of regulated exocytosis. The intracellular Ca 2ϩ concentration and membrane capacitance were then recorded simultaneously over the following 8 min. Note the rapid but transient increase in intracellular Ca 2ϩ which is followed by a slower but sustained increase in membrane capacitance.

FIG. 3. Liposome binding of annexin II-p11 in the presence or absence of N-terminal annexin II peptides.
Phospholipid liposomes were mixed with monomeric annexin II expressed in SF9 insect cells (panel 1) or with the heterotetrameric annexin II-p11 complex purified from intestinal epithelium (panels 2-4). In panels 3 and 4 the complex was pretreated with a 500-fold molar excess of the N-terminal annexin II peptides N1-14 (panel 3) or Ac1-14 (panel 4) and the respective peptides remained present throughout the phospholipid binding assay. The mixtures were incubated for 30 min and liposome bound proteins (lanes P) were then separated from the unbound fraction (lanes S) by centrifugation. Identical aliquots of the different fractions were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with antibodies directed against annexin II and p11, respectively. Note the Ac1-14 induced disruption of the annexin II-p11 complex which leads to a liberation of the majority of p11 from the liposome pellet. In contrast, no complex disruption is observed in the presence of the non-acetylated peptide N1-14. The nonbound annexin II seen in lanes S of panels 2-4 represents monomeric protein present in the complex preparation from intestinal epithelium. The annexin II retained in the liposome pellet (lane P) of panel 4 most likely is a mixture of two species. First, some residual p11-complexed annexin II since a faint p11 signal is seen in this lane on overexposed blots. Second, monomeric annexin II which is bound to liposomes aggregated due to the action of the residual annexin II-p11 complexes present. In contrast, non-aggregated liposomes fail to bind monomeric annexin II under the conditions chosen (panel 1).

The Annexin II-p11 Complex Is Involved in Regulated Exocytosis in Endothelial Cells-Annexin II has been localized to the subplasmalemmal region of different types of cells, and this
localization is thought to be of crucial importance for a role of the protein in Ca 2ϩ -regulated secretion in adrenal chromaffin cells (11). The tight association of annexin II with the cell cortex depends on its complex formation with p11 (11,32), and this is mediated through the N-terminal domain of annexin II (33,34). The N-terminal 14 residues of annexin II (AcSTVHEI-LCKLSLEG) including the N-acetyl group of the N-terminal serine harbor the entire p11 binding site, and synthetic peptides comprising this sequence (herein referred to as Ac1-14) bind p11 with high specificity and affinity. In contrast, peptides of the same sequence containing a nonmodified N terminus (N1- 14) show a more than 1000-fold reduced affinity for p11 (6,7).
Based on the high affinity of the Ac1-14/p11 interaction we analyzed whether the peptide in addition to competing with annexin II for p11 binding (6) could also disrupt preformed and lipid bound annexin II-p11 complexes and therefore could be used as a function-interfering reagent. Liposome pelleting as-says were performed to elucidate whether Ac1-14 is capable of liberating p11 from liposome-bound annexin II-p11 complexes. Experiments were carried out at a Ca 2ϩ concentration of 250 nM, i.e. at a level experienced in the GTP␥S-stimulated endothelial cells (see Fig. 2). Under these conditions monomeric annexin II, which was expressed recombinantly in SF9 insect cells, fails to bind to the phospholipid liposomes, whereas the annexin II-p11 complex purified from porcine intestinal epithelium effectively interacts with the phospholipids and is copelleted together with the liposomes by centrifugation (Fig. 3). This behavior of the complex is not affected by inclusion of a 500-fold molar access of the nonacetylated peptide N1-14, i.e. the sequence encompassing the N-terminal 14 residues of annexin II with a nonmodified N-terminal end. In contrast, the same amount of the acetylated peptide Ac1-14 significantly interferes with liposome binding of annexin II-p11. Fig. 3 shows that preincubation with the Ac1-14 peptide effectively disrupts the complex resulting in a liberation from the liposome bound fraction of p11 which itself is incapable of binding phospholipids (not shown). Thus, the Ac1-14 peptide but not its non-acetylated FIG. 4. GTP␥S induced changes in membrane capacitance in the presence or absence of N-terminal annexin II peptides and the Ca 2؉ chelator BAPTA. A, CPAE cells were patched and 100 M GTP␥S was added to the pipette solution to induce secretion. GTP␥S was omitted in the patched cell shown as a negative control. As indicated the individual cells were either treated with GTP␥S alone or with GTP␥S in the presence of BAPTA (10 mM) or the N-terminal annexin II peptides Ac1-14 or N1-14, respectively. Note that the GTP␥S-induced capacitance increase is unaffected in cells loaded with N1-14, whereas the annexin II-p11 complex-disrupting peptide Ac1-14 reduces the capacitance increase by more than 50%. B, summary of capacitance measurements in the presence or absence of N1-14, Ac1-14, and BAPTA, respectively. The total number of cells analyzed and the standard errors are indicated. counterpart can dissociate preexisting annexin II-p11 complexes and will therefore interfere with phospholipid binding and vesicle aggregating properties displayed by the complex.
Given the complex-disrupting properties of Ac1-14 and the proposed involvement of annexin II-p11 in Ca 2ϩ -triggered secretion, we analyzed the effect of the peptide on regulated exocytosis in CPAE cells. Ac1-14 or N1-14 (as a control peptide) were given together with GTP␥S, and the secretory responses in the respectively loaded cells were measured by tracking membrane capacitance. Only cells responding to the GTP␥S stimulus with a transient Ca 2ϩ increase were considered in this analysis. The individual cells were loaded with 1 mM peptide to achieve an approximately 1000-fold molar excess over endogenous annexin II whose concentration in CPAE cells was calculated by semiquantitative Western blots to be approximately 1 M (22). Fig. 4A shows that the nonacetylated control peptide has no effect on the GTP␥S-induced capacitance increase. The presence of Ac1-14, however, markedly reduces the exocytotic response. Independent tracings of a number of individually loaded cells reveal that the reduction in capacitance increase elicited by Ac1-14 is approximately 50% and that the Ac1-14 effect is highly specific, as no reduction in GTP␥Sinduced capacitance increase is observed in the presence of N1-14 (Fig. 4B). To analyze whether the Ac1-14 peptide is acting on a Ca 2ϩ -dependent system or on a Ca 2ϩ -independent system activated by GTP␥S, we performed membrane capacitance studies on cells loaded with the Ca 2ϩ chelator BAPTA. The results show that buffering intracellular Ca 2ϩ at a low concentration results in a strongly reduced capacitance increase upon GTP␥S treatment (Fig. 4, A and B). This indicates that the Ac1-14 peptide inhibits only the Ca 2ϩ -dependent exocytosis activated by GTP␥S.
Based on the Ca 2ϩ -regulated phospholipid binding properties of annexin II-p11, which are displayed at Ca 2ϩ concentrations found in GTP␥S-activated CPAE cells, it seems likely that the complex acts at the level of secretory vesicles and/or at the level of the plasma membrane possibly by linking vesicles to one another or to the plasma membrane. To distinguish between these possibilities we analyzed in detail the intracellular localization of annexin II in CPAE cells and compared this to a typical secretory vesicle marker, vWF. Fig. 5A shows that CPAE-annexin II is mainly plasma membrane-associated, although a fraction of the protein appears to reside on intracellular structures, probably resembling elements of the endosomal compartment (as previously shown in baby hamster kidney and Madin-Darby canine kidney cells (35)(36)(37)). An indistinguishable staining pattern is observed for p11 (not shown), indicating that the membrane-associated annexin II resides in a complex with p11. vWF, on the other hand, is almost exclusively found in intracellular structures (see also Fig. 1) which resemble Weibel-Palade bodies and show no or only very little overlap with the annexin II/p11 positive elements (Fig. 5B). The predominant localization of annexin II at the plasma membrane, which clearly differs from the vWF staining, is also illustrated by a confocal Z scan of the annexin II labeling (Fig.  5C). Thus, in the process of regulated exocytosis, annexin II-p11 most likely acts close to or at the plasma membrane. DISCUSSION In this report we establish for the first time membrane capacitance tracing as an approach to the study of regulated exocytosis in endothelial cells at the single cell level. We show that a substantial increase in cell surface area indicative of secretory vesicle fusion with the plasma membrane occurs in GTP␥S-stimulated endothelial cells and that in the majority of the cells this increase is preceded by a transient rise in intracellular Ca 2ϩ . This secretory response most likely involves Weibel-Palade bodies, the principal storage granules for von Willebrand factor and P-selectin in endothelial cells. vWFpositive structures are present in the CPAE cells employed (Fig. 1) and immunofluorescence analysis of nonpermeabilized endothelial cells reveals that the internally stored vWF is released to the cell surface once the intracellular Ca 2ϩ concentration is elevated through the action of the Ca 2ϩ ionophore A23187 (data not shown). However, other populations of endothelial storage granules may also contribute to the exocytotic response observed after GTP␥S infusion and subsequent Ca 2ϩ elevation. These could include storage particles for endothelin-1 and tissue-type plasminogen activator, which have been identified recently and which are morphologically and biochemically distinct from vWF-positive Weibel-Palade bodies (38,39).
The capacitance measurements in combination with the use of a peptide disrupting the annexin II-p11 complex also show for the first time at the single cell level that the complex is involved in regulated exocytosis. Previously, various lines of biochemical evidence as well as data from experiments using digitonin-permeabilized cells have implicated the heterotetrameric annexin II-p11 complex in Ca 2ϩ -regulated secretion in adrenal chromaffin cells (2)(3)(4). The complex, but not monomeric annexin II, is capable of aggregating chromaffin granules at Ca 2ϩ concentrations occurring in stimulated cells. In such simple protein-granule mixtures annexin II-p11 following vesicle aggregation is even able to display fusogenic properties, however, only in the presence of elevated concentrations of cis-unsaturated fatty acids (8). Moreover, annexin II has been identified as a component which is lost from chromaffin cells upon digitonin permeabilization and upon adding back can retard the run-down in Ca 2ϩ -triggered secretion observed in the permeabilized cells (12,13). This effect of annexin II appears to require complex formation with p11 and phosphorylation by protein kinase C, an enzyme stimulated in activated chromaffin cells (13). Interestingly, the proposed function of annexin II-p11 in chromaffin granule exocytosis correlates with a translocation of annexin II from the cytosol to the submembraneous region observed following nicotinic stimulation of chromaffin cells (11). Such a translocation might also occur in activated endothelial cells although here the majority of annexin II (and p11) is already found in the cortical region of nonstimulated cells (Fig. 5C). Based on these immunocyto- chemical data it appears likely that in endothelial cell exocytosis annexin II-p11 functions in the submembraneous region, possibly by anchoring secretory vesicles in the cell cortex.
Although the mechanistic basis of the annexin II-p11 function in regulated exocytosis is not known several morphological observations point to a structural role in linking vesicles to the plasma membrane and/or the cortical cytoskeleton. An ultrastructural analysis of stimulated chromaffin cells, for example, located annexin II between the plasma membrane and the facing chromaffin granule membranes, possibly forming fine strands cross-linking the opposing membranes (14). A high resolution image of intermembrane junctions formed by annexin II-p11 was obtained recently using cryo-electron microscopy of artificial liposomes and chromaffin granules aggregated by annexin II-p11 in a Ca 2ϩ -dependent manner. The junctions observed were of constant thickness (210 Å) and showed a symmetric distribution of electron-dense material, most likely representing annexin II molecules bound to the outer leaflets of each vesicle and linked via a central p11 dimer (40). A structural role of the annexin II-p11 complex is also in line with the relative abundance of the proteins in cells where they have now been implicated in regulated secretion, i.e. chromaffin and endothelial cells. It remains to be seen whether and how annexin II-p11, as an entity linking secretory vesicle to the cell cortex, cooperates with the SNARE-based docking and fusion machinery (41) in regulated exocytosis. In contrast to the SNARE complexes, annexin II-p11 is probably not an obligatory component of the exocytotic machinery since it is not expressed to similarly high levels in all cells capable of regulated secretion and since in PC 12 cells expression of a trans-dominant annexin II-p11 mutant does not interfere with dopamine secretion triggered by Ca 2ϩ elevation in digitonin-permeabilized cells (15). Interestingly, application of the Ac1-14 peptide disrupting the annexin II-p11 complex does not result in a complete inhibition of the exocytotic response in CPAE cells. This could mean that in vivo peptide-mediated complex disruption is not complete, e.g. because a portion of annexin II-p11 is incorporated into higher order structures and thereby protected from the peptide action. Alternatively, it remains possible that the different pathways of regulated exocytosis most likely existing in endothelial cells (see above) depend differently on annexin II-p11.
In addition to Ca 2ϩ , phosphorylation by protein kinase C most likely represents another signal regulating annexin II-p11 activities during Ca 2ϩ -triggered secretion. Chromaffin cell annexin II is phosphorylated by PKC in response to nicotinic stimulation (42), and only phosphorylated annexin II-p11 is able to restore secretion in permeabilized chromaffin cells depleted of their PKC activity (13). Moreover, in adrenergic chromaffin cells a synthetic annexin II peptide corresponding to a sequence in the N-terminal domain, which includes the major PKC acceptor site, Ser-25, inhibits both nicotine-induced recruitment of annexin II to the cell periphery and nicotinetriggered exocytosis (11). While the mechanistic consequences of the regulatory PKC phosphorylation in living cells are not known, it is interesting to note that phosphorylation by PKC of chromaffin granule-bound annexin II induces a fusion of the granule membranes (10). In addition, PKC phosphorylation of annexin II-p11 inhibits the vesicle aggregation properties displayed by the complex (43). Finally, PKC phosphorylation of monomeric annexin II at a second site, Ser-11, inhibits p11 binding and thus formation of the heterotetrameric annexin II-p11 complex (9,18). These biochemical analyses indicate that the annexin II function is subject to multiple regulations, which could differ from cell type to cell type. In chromaffin cells activation of PKC, e.g. by nicotinic stimulation, probably in-duces a translocation of (phosphorylated) annexin II to the submembraneous region, whereas annexin II is already concentrated in the periphery of nonstimulated endothelial cells (Fig. 5). Complex formation with p11, which itself can be finetuned by PKC action, establishes the heterotetrameric entity capable of linking membrane surfaces to one another and/or to an underlying cytoskeleton in a Ca 2ϩ -regulated manner. The experimental data obtained here show for the first time that the integrity of this annexin II-p11 complex with its crosslinking activity is indispensable for efficient regulated exocytosis in endothelial cells.