Fusion Of Lamellar Bodies Research Articles

Calcium-Dependent Self-Association of Annexin II: A Possible Implication in Exocytosis

Alveolar type II cells secrete lung surfactant through exocytosis of lamellar bodies. We previously showed that the annexin II tetramer (Anx IIt) mediates the fusion of lamellar bodies with liposomes. The present study examined the possible involvement of membrane proteins in this process. Pre-treatment of lamellar bodies with trypsin and α-chymotrypsin reduced Anx IIt-mediated membrane fusion. With the use of an Anx IIt-conjugated Sepharose column, three Anx IIt-binding proteins with molecular weights of 67,000, 36,000 and 34,000 were isolated from the Triton X-100 extract of bovine lung tissue membranes. These proteins were identified as annexins VI, II and IV by Western blot. The interaction of Anx IIt with annexins II and IV was confirmed by ligand blot assay. An EGTA-resistant membrane-bound annexin II was present in lung type II cells. Anx IIt preferentially bound to membranous annexin II compared with cytosolic annexin II of type II cells. With the use of immunofluorescence, annexin II was found to translocate from cytoplasm to plasma membranes in type II cells upon stimulation with phorbol 12-myristate 13-acetate. These results suggest that cytosolic annexin II may bind to membranous annexin II and form a protein–protein bridge to bring two membranes together.

The secretory granular cell: the outermost granular cell as a specialized secretory cell.

The contents of epidermal lamellar bodies (LB) are delivered selectively to the intercellular spaces at the stratum granulosum (SG)-stratum corneum (SC) interface. We assessed the subcellular basis for LB secretion first by confocal microscopy, following labeling with Nile red or NBD-ceramide, which reveals a tubulo-reticular membrane system within the apical cytosol of the outermost SG cell layer under basal conditions, changing to a more peripheral staining pattern when secretion is stimulated. Ultrastructural study demonstrates that this network is composed of a widely disbursed trans-Golgi-like network (TGN), associated with arrays of contiguous LB, and deep invaginations of the SG-SC interface. Under basal conditions, limited fusion of apically directed LB leads to deep, interconnected invaginations of the apical plasma membrane, resulting in the formation of an extensive, honeycomb extension of the SG-SC interface. Still deeper invaginations and more extensive organelle fusion develop after the epidermis is acutely permeabilized by either acetone treatment, sonophoresis, or iontophoresis. Finally, nascent LB appear to bud off cisternae of the TGN, a process that appears to accelerate after barrier disruption. The deep invaginations of the SG-SC interface; the wide distribution of the TGN within the apical cytosol; the association of nascent LB with the TGN; and the rapid fusion of LB with these invaginations, deep within the cytosol, account for (i) the polarized secretion of LB from the apex of the outermost SG cell, and (ii) the rapid LB-secretory response to barrier perturbations. Finally, our results point to the outermost SG cell as a uniquely specialized secretory cell. We propose the term "secretory granulocyte" to encompass the specialized features of these cells.

Early alterations in intracellular and alveolar surfactant of the rat lung in response to endotoxin.

The aim of this study was to characterize early ultrastructural, biochemical, and functional alterations of the pulmonary surfactant system induced by Salmonella minnesota lipopolysaccharide (LPS) in rat lungs. Experimental groups were: (1) control in vitro, 150 min perfusion; (2) LPS in vitro, 150 min perfusion, infusion of 50 microg/ml LPS after 40 min; (3) control ex vivo, 10 min perfusion; (4) LPS ex vivo, lungs perfused for 10 min from rats treated for 110 min with 20 mg/kg LPS intraperitoneally. Morphometry of type II pneumocytes showed that LPS increased stored surfactant. Lamellar bodies were increased in size, but decreased in numerical density, suggesting that giant lamellar bodies observed in LPS-treated lungs may result from fusion of normal bodies. Structural analysis of alveolar surfactant composition showed that LPS elicited an increase in lamellar body-like and multilamellar forms. Bronchoalveolar lavage (BAL) material from LPS-treated lungs was decreased in phospholipids. BAL bubble surfactometer analysis showed a reduction in hysteresis area caused by LPS. We conclude that LPS leads to alterations of intracellular and alveolar surfactant within 2 h: fusion of lamellar bodies, reduction in surfactant secretion, and changes in alveolar surfactant transformation, composition, and function, which may contribute to the development of respiratory distress.

Dynamics of surfactant release in alveolar type II cells.

Pulmonary surfactant, secreted via exocytosis of lamellar bodies (LB) by alveolar type II (AT II) cells, maintains low alveolar surface tension and is therefore essential for normal lung function. Here we describe real-time monitoring of exocytotic activity in these cells by visualizing and quantifying LB fusion with the plasma membrane (PM). Two approaches were used. First, fluorescence of LysoTracker Green DND-26 (LTG) in LB disappeared when the dye was released after exocytosis. Second, phospholipid staining by FM 1-43 resulted in bright fluorescence when this dye entered the LB through the fusion pore. Both processes were restricted to and colocalized with LB and occurred simultaneously. In AT II cells, FM 1-43 offered the unique advantage to independently define the moment and cellular location of single exocytotic events as well as the amount of material released, and to monitor its extracellular fate. Furthermore, both dyes could be used in combination with fura-2. The results indicate considerable diversity in the dynamics of LB exocytosis. In the majority of cells stimulated with ATP and isoproterenol, the first fusion of LB coincided with the rise of [Ca2+]i, but subsequent response of other LB in the same cell considerably outlasted this signal. In other cells, however, the onset of exocytosis was delayed by several minutes. After LB fusion, release of surfactant from LB into an aqueous solution was slow. In summary, stimulated exocytosis in AT II cells occurs at a much slower rate than in most other secretory cells but is still a more dynamic process than predicted from conventional measurements of surfactant released into cell supernatants.

Calcium-dependence of synexin binding may determine aggregation and fusion of lamellar bodies.

Synexin (annexin VII) is a member of the annexin family of calcium and phospholipid binding proteins that promote calcium-dependent aggregation and fusion of lipid vesicles or secretory granules. We have previously suggested that synexin may be involved in membrane fusion processes during exocytosis of lung surfactant since it promotes fusion in vitro of lamellar bodies with plasma membranes. In this study, we characterized calcium-dependency of synexin binding to lamellar bodies and plasma membranes, since such binding is the initial, and, therefore, may be the rate-limiting step in membrane aggregation and fusion. The binding of biotinylated synexin to lamellar bodies and plasma membranes increased in a calcium-dependent manner reaching a maximum at approx. 200 microM Ca2+. Binding to lamellar bodies was completely inhibited by unlabelled synexin. Gel-overlay analysis showed that synexin bound to an approx. 76 kDa protein in the lamellar body and plasma membrane fractions. The calcium kinetics were noticeably similar for synexin binding to lamellar bodies and plasma membranes, aggregation of lamellar bodies, and fusion of lamellar bodies with lipid vesicles. At low calcium concentrations, aggregation of lamellar bodies could be increased with increasing synexin concentration, and arachidonic acid increased all three parameters (binding, aggregation, and fusion) in a similar manner. The effects of calcium and arachidonic acid on these three parameters suggest that synexin binding to lamellar bodies may be a rate-determining step for fusion during surfactant secretion. Furthermore, at near physiological calcium levels, the membrane fusion may be enhanced by elevated concentrations of synexin and polyunsaturated fatty acids.

Ultrastructural changes of lung capillary endothelium in response to botulinum C2 toxin.

The role of the endothelial cytoskeleton for the structural integrity of the pulmonary gas exchange area was probed with the use of Clostridium botulinum C2 toxin. This agent causes selective loss of nonmuscle F-actin. In buffer-perfused rabbit lungs, vascular pressures were kept within physiological ranges. In different groups, low-dose [0.3 (C2,I)/0.6 (C2,II) ng/ml] and high-dose [10 (C2,I)/20 (C2,II) ng/ml] toxin were applicated into the buffer fluid; experiments were terminated after a total weight gain of either 1 or 7.5 g. Electron microscopy revealed extensive attenuations, undulations, and protrusions of the endothelial layer, suggestive of "remodeling" and "flowing" of the cell membrane in low C2 toxin-treated lungs accompanied by few disruptions of the endothelial layer and edema formation. In addition, endothelial cells displayed vesiculation and bleb formation. Lungs that were exposed to high-toxin doses displayed marked attenuations of the endothelial layer in addition to large endothelial cell disruptions, which did not include interendothelial junctions. Interestingly, type II epithelial cells displayed fusion of lamellar bodies. Collectively, these data suggest that the actin microfilament system is instrumental in supporting endothelial cell membrane configuration and integrity and maintains the intimal barrier function of the lung microvasculature.

Lung annexin II promotes fusion of isolated lamellar bodies with liposomes

The role of annexin II in the secretion of lung surfactant was investigated using isolated lamellar bodies and/or liposomes as the model system for aggregation and fusion. We first compared membrane aggregation mediated by two forms of annexin II, annexin II monomer (Anx IIm) and annexin II tetramer (Anx IIt). Anx Ill required 20-fold less Ca 2+ to mediate phosphatidylserine (PS) liposome aggregation compared to Anx IIm. Aggregation of lamellar bodies mediated by Anx IIt was 4-fold greater than that by Anx IIm at 1 mM Ca 2+. These results suggest that Anx IIt may be the more active form in vivo. Fusion of lamellar bodies with PS liposomes was promoted by Anx IIt in a dose-dependent manner, with maximal fusion occurring at 10–15 μg/ml of Anx IIt. Fusion was dependent on Ca 2+ and the phospholipid composition of liposomes. While the fusion of lamellar bodies with PS liposomes required 100 μM Ca 2+, the fusion with PS/phosphatidylethanolamine (PE) (I:3) liposomes required only 10 μM Ca 2+. Anx IIt-mediated lamellar body-liposome fusion was enhanced by arachidonic acid, a lung surfactant secretagogue and inhibited by 4.4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of lung surfactant secretion. The data suggest that Anx IIt may play a role in the fusion of lamellar bodies with plasma membranes during lung surfactant secretion.

Stilbene disulfonic acids inhibit synexin-mediated membrane aggregation and fusion

Stilbene disulfonic acids inhibit surfactant secretion from lung epithelial type II cells by an undefined mechanism, and inhibit CD4 mediated cell-cell fusion. We have previously shown that lung synexin promotes in vitro fusion of lamellar bodies and plasma membranes, an obligatory process for surfactant secretion. This study investigates the effect of stilbene disulfonic acids, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), and 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMDS), on synexin-mediated liposome aggregation and fusion. Structurally, these three stilbene compounds differ in the number of isothiocyano groups present (DIDS = 2, SITS = 1, and AMDS = 0). At 10 μg synexin/ml, DIDS and SITS inhibited synexin-mediated liposome aggregation with an EC 50 of 3.5 μM and 148 μM, respectively. In comparison, AMDS was least inhibitory (EC 50 > 1 mM). Thus, the inhibitory potency (DIDS > SITS > AMDS) was partly dependent upon the number of isothiocyano groups. The EC 50 was also dependent on synexin concentration. Stilbene disulfonic acids were also inhibitory for arachidonic acid-enhanced synexin-mediated liposome fusion. The EC 50 for DIDS and SITS for fusion were similar to that for liposome aggregation. Ca 2+-induced synexin polymerization, measured by 90° light scattering, was increased by DIDS, suggesting binding of stilbene disulfonic acids to synexin. The binding of DIDS to synexin was dependent on the molar ratio of synexin to DIDS. These results indicate that stilbene disulfonic acids interact directly with synexin to inhibit membrane aggregation and fusion. Our results suggest that such inhibition of synexin activity may contribute towards inhibition of surfactant secretion by DIDS, and support a physiological role for synexin in lung surfactant secretion.

Regulation of lung surfactant secretion

Secretion of lung surfactant is the direct step in release of the lipoprotein-like product, synthesized in lung epithelial type II cells, onto the alveolar surface. Release of surfactant phosphatidylcholine (PC) proceeds via formation of surface pores during exocytosis of lamellar bodies. Surfactant secretion is regulated locally in the lung by changes in ventilation rate, possibly mediated by distension and altered intracellular pH. Secretion is also stimulated by various agents, including agonists for beta-adrenergic, purinoceptors, and vasopressin receptors and is associated with increased cytosolic Ca2+, cellular adenosine 3',5'-cyclic monophosphate, and activation of protein kinases. Limited studies suggest that secretion of surfactant protein A may be regulated by both cAMP-dependent and protein kinase C-dependent pathways. The integration of these various mechanisms for the in vivo regulation of surfactant secretion remains largely unexplored. Future research into the mechanisms involved in lamellar body fusion with the plasma membrane, role of protein phosphorylation, transient changes in cAMP and Ca2+, and coordination between the secretion of phospholipid and protein components of surfactant should enhance our understanding of secretion of surfactant "lipoprotein."

THE ROLE OF CYCLIC AMP IN LAMELLAR BODY SYNTHESIS & SECRETION

To assess the effect of dibutryl cyclic 3′-5′ adenosine monophosphate (DBcAMP) on some aspects of pulmonary Type II cell morphology and biochemistry, adult rats were injected i.p. with 47 mg/kg DBcAMP and sacrificed at intervals. Control rats received saline or 5′ AMP. Lung tissue was prepared for electron microscopy. 5 min. postinjection, Type II cells in treated rats appeared more numerous than in controls and endoplasmic reticulum (ER) dilated. By 10 min. the cells appeared larger, lamellar bodies (LB) were increased per cell and ER remained dilated. Multivesicular bodies (MVB) appeared in increased numbers. By 15 min. the cells were swollen, often with loss of microvilli and MVB's conspicuous. There appeared to be fusion of LB limiting membranes with plasma membranes and extrusion of LB contents into alveoli. By 30 min. macrophages appeared in alveoli and septa. Type II cells were of more normal size and vacuoles appeared in the cytoplasm near the alveolar surface suggestive of endocytosis. By 60 min. Type II cells were not remarkable. Choline kinase (CK) activity was assayed in LB rich cell fractions from pooled lung of 6 DBcAMP treated and 6 control rats sacrificed at 15 min. There was a 5-fold increase in CK activity in the DBcAMP treated rats. These findings suggest that intracellular synthesis and extrusion of LB from Type II cells may be mediated by cAMP.