review cellular physiology · 2009-11-27 · 2 provided by electron microscopy (em) studies [10]....

1

Review

Cell Physiol Biochem 2010;25:01-12 Accepted: November 09, 2009Cellular PhysiologyCellular PhysiologyCellular PhysiologyCellular PhysiologyCellular Physiologyand Biochemistrand Biochemistrand Biochemistrand Biochemistrand Biochemistryyyyy

Copyright © 2010 S. Karger AG, Basel

Fax +41 61 306 12 34E-Mail [email protected]

© 2010 S. Karger AG, Basel1015-8987/10/0251-0001$26.00/0

Accessible online at:www.karger.com/cpb

Lamellar Body Exocytosis by Cell Stretch orPurinergic Stimulation: Possible PhysiologicalRoles, Messengers and Mechanisms

Paul Dietl1, Birgit Liss1, Edward Felder1, Pika Miklavc1 and HubertWirtz2

1Institute of General Physiology, University of Ulm, Ulm, 2Department of Respiratory Medicine, UniversityClinic Leipzig, Leipzig

Paul DietlInstitute of General Physiology, University of UlmAlbert-Einstein-Allee 11, 89081 Ulm (Germany)Tel. 0049-731-50023231, Fax 0049-731-50023242E-Mail [email protected]

Key WordsExocytosis • Fusion • Hemifusion • Surfactant •Alveolus • Lung • Type II cell • Stretch •Mechanotransduction • ATP

AbstractA major function of the pulmonary alveolar type II cellis the secretion of surfactant, a lipoprotein-like sub-stance, via exocytosis of secretory vesicles termedlamellar bodies (LBs). The process of surfactant se-cretion is remarkable in several aspects, consideringstimulus-delayed fusion activity, poor solubility of vesi-cle contents, long hemifusion lifetimes, slow fusionpore expansion and active, actin-driven content re-lease. Cell stretch as well as P2Y2 receptor stimula-tion by extracellular ATP are considered the mostpotent stimuli for LB exocytosis. For both stimuli, el-evation of the cytoplasmic Ca2+ concentration [Ca2+]cis a key step. This review summarizes possible physi-ological roles and pathways of stretch- or ATP-in-duced surfactant secretion and discusses molecularmechanisms controlling the pre-, hemi- andpostfusion phase, in comparison with neuroendocrinerelease mechanisms.

Introduction

Surfactant, short for “surface active agent”, is alipid-rich, lipoprotein-like substance, secreted into thelumen of the pulmonary alveolus. It consists of mainlyphospholipids and four specific surfactant proteins (SP)[1, 2]. Surfactant is a crucial secretory product duringand after birth, its continuous and accurate secretion andfunction is indispensable throughout life. The discoveryof surface tension as the major component of retractiveforces in the lung was made as early as 1929 [3], but itwas not until the 1950s and early 1960s that activesurface material from the lung was isolated and charac-terized [4, 5]. Its deficiency causes infant respiratory dis-tress syndrome (IRDS) [6]. The lamellar body (LB) withinthe type II pneumocyte was identified as the intracellularvesicular storage site of surfactant [7]. The LB is a largeorganelle (about 1-2 µm Ø) that stores surfactant in anextremely compact, lamellar conformation (recentlyreviewed in [8]). Although the number of type IIpneumocytes is about twice the number of type Ipneumocytes, they represent only about 7% of thealveolar surface [9]. The first convincing evidence in favorof an exocytotic surfactant release mechanism was

2

provided by electron microscopy (EM) studies [10].The phospholipid composition of LBs isolated from typeII pneumocytes is similar to that of whole lung surfactantobtained from broncho-alveolar lavage (BAL) [11, 12].Essentially all alveolar surfactant phospholipids are se-creted via exocytosis of LBs [13]. In addition to thephospholipids, all four surfactant proteins (SP-A, SP-B,SP-C, and SP-D) which account for about 10% by weight,can be obtained by BAL [8, 14]. Their respective distri-butions within LBs and BAL are different: The smallhydrophobic SP-B and SP-C are localized within LBsand co-secreted with all other LB contents. Both pro-teins are believed to play an important role in squeezingout non-dipalmitoyl phosphatidylcholine (DPPC) compo-nents during film formation and compression at the air-liquid-interface, which results in a highly DPPC-enrichedsurface film [8, 15]. In contrast, the large hydrophilicSP-A and SP-D are secreted largely independently ofLB contents by other routes. Although SP-A has an in-hibitory effect on LB exocytosis [16], these proteinsappear to be mainly involved in pulmonary host defense.Surfactant secretion has been extensively reviewed else-where [17-21]. This review focuses on the regulation ofthe exocytotic process of LBs by the two probably mostpotent and physiologically important stimuli: alveolarstretch or extracellular ATP. In particular, we shalldiscuss and distinguish between mechanisms before andafter fusion of the LB with the plasma membrane, incomparison with neuroendocrine release mechanisms.

Ventilation and mechanical effects on al-veolar epithelial cell shape

It has been known for decades that large lung infla-tions stimulate surfactant secretion [22-27]. Whenthe lung is distended during inspiration, at least two dif-ferent forms of mechanical stress (defined as force perunit of area) can act on the epithelium of the respiratoryzone:

1. Re-opening of collapsed airways can induce shearstress on the epithelium and connective tissue. This shearstress may play an important role for ventilator-inducedlung injury during artificial ventilation of diseased lungs(reviewed in detail in [28]).

2. Probably even more relevant is the fact that al-veoli are subject to a tensile strain (= stretch, defined asa change in length in relation to the initial length).Under the assumption of the alveolus as a homogenouslyinflated balloon, each spot should be stretched in an

equi-biaxial way, i.e. by the same length in all directions.However, this strain model is likely a gross simplification,due to the uneven alveolar shape and an alveolar wallthat is probably not homogenous in its elastic properties[29], resulting in an unisotropic stress distribution [30, 31].In fact, it is still a matter of debate how much inflation isactually needed to stretch individual epithelial cells afterunfolding of connective tissue (analogous to theextension of an accordion). From alveolar surface to vol-ume relationships, Gil and Weibel concluded that alveolarsurface changes are primarily due to folding and unfold-ing of septa before plastic tissue changes occur [32].On the other hand, a morphometric analysis byTschumperlin and Margulies revealed significant changesof the epithelial basement membrane surface areaabove 40% of total lung capacity [33], indicatingepithelial cell deformation at physiologically relevant lungvolumes.

Stretch-induced signalling of alveolar typeII cells

Live cell imaging of single cells during static or cy-clic stretch is a methodological challenge, and the eluci-dation of stretch-induced Ca2+ signals with high temporaland special resolution is just beginning to emerge [34].Alveolar type II cells in culture (mostly isolated from ratlungs) are very sensitive to tensile strain and respond to asingle stretch - increasing the cell surface area only byabout 10 % - with an elevation of the cytoplasmic Ca2+

concentration [Ca2+]c and stimulated surfactant secre-tion [35, 36]. Thus, it is likely, that the increase of sur-factant secretion, which is observed after only a singleinflation of the isolated perfused lung [25], is the result ofa related epithelial cell stretch. Recently however, thenotion of the type II pneumocyte as the alveolar stretchsensor was challenged by an in situ study, based onfluorimetric measurements of [Ca2+]c [37], suggesting thattype I pneumocytes are the primary responders to alveo-lar inflation. Using gap junction blockers, Ashino andcollegues concluded that Ca2+ signals can promote fromtype I cells to type II cells, consistent with the notion thatsmall signalling molecules can permeate gap junctions [38].A distinct but related mechanism was recently proposedusing mechanically stretched co-cultures of type I andtype II rat pneumocytes: These data suggest that alveo-lar type I cells act as mechanosensors, releasing ATP inresponse to stretch activation, which subsequentlystimulates type II cells in a paracrine way [39].

Dietl/Liss/Felder/Miklavc/WirtzCell Physiol Biochem 2010;25:01-12

3

All these models, however, converge in an elevationof [Ca2+]c in the type II pneumocyte, where surfactant isstored and released by LBs. Ca2+ entry from theextracellular space and Ca2+ release from intracellularstores both contribute to this [Ca2+]c elevation [35, 36].Ca2+ entry from the extracellular space is indeed essen-tial for surfactant release from stretched type II cells,which renders mechano-sensitive ion channels asparticularly promising candidates [35]. Nevertheless, themolecular basis for the stretch-induced Ca2+ entry - inparticular the molecular entities of stretch-induced Ca2+

channels - are still obscure. It should be noted thatdepolarizing membrane potentials do not result in Ca2+

entry into type II pneumocytes, because those cells donot express functional voltage-dependent Ca2+ channels(CaV) [40]. Thus, the entry of extracellular Ca2+ throughCaV is not possible, which is a fundamental difference toneuroendocrine secretion, where CaV is an importantmediator in excitation-secretion-coupling. Consequently,in contrast to neuroendocrine secretion, non-selectivecation channels can only be considered to contribute tothe rise of [Ca2+]c if they are permeable for Ca2+ [41],since indirect activation via voltage-gated Ca2+ entry isabsent. This difference between epithelial and neuroen-docrine secretion is illustrated in Fig. 1.

Among Ca2+ permeable channels which mightbe involved in stretch-induced Ca2+ entry in the type IIcell, three different functional categories comeinto consideration (Fig. 1):

Mechanosensitive channels (MSC), store-operatedchannels (SOC), and second messenger-operated

channels (SMOC).1. Mechanosensitive channels (MSC): Recently,

several Ca2+ permissive members of the transientreceptor potential (TRP) channel family have beenproposed as stretch-sensitive channels; and channels ofthe epithelial Na+ channel (ENaC)/degenerin family areother candidates [42-49]. Tissue specific mRNA expres-sion analysis (RT-PCR and in situ hybridization) suggestseveral potentially mechanosensitive TRP channels of theV and C subfamilies in the lung as candidates, but func-tional evidence is yet lacking [50].

2. Store-operated channels (SOC): Since cellstretch in the type II cell causes Ca2+ release fromthapsigargin-sensitive intracellular stores [35, 36], theactivation of a store-operated Ca2+ entry pathway mustbe taken into consideration. Thapsigargin is an inhibitorof the endoplasmic Ca2+ ATPase and frequently used asa pharmacological tool to specifically stimulate SOC inthe absence of phosphoinositide breakdown [51]. Althoughdifferent conductances and ion selectivities of SOC havebeen presented , a highly Ca2+ selective channel termedCRAC (Ca2+ release activated Ca2+) channel is bestcharacterized [52]. CRAC channel activation involvesphysical migration of endoplasmic reticulum (ER)-resi-dent STIM proteins to ER–plasma membrane junctionsand subsequent aggregation of the Ca2+ influx channelOrai (recently reviewed by [53]). To this end, the exist-ence and potential physiological role of CRAC channelsin type II cells remains to be elucidated.

3. Second messenger-operated channels (SMOC).The impact of static or cyclic stretch on extracellular matrix

Fig. 1. Stimulus-secretion-coupling in neuroendocrinecells (left) and epithelial (type II) cells (right). Inneuroendocrine cells, a depolarization of themembrane potential (e.g. due to K+ channel block orcation channel activation) triggers the activation ofvoltage-dependent Ca2+ channels (CaV), which leadsto Ca2+ entry and subsequent fusion of secretory vesi-cles (green) with the plasma membrane within millisec-onds. In type II epithelial cells, the resting membranepotential is part of the electrochemical force, whichdrives Ca2+ ion influx presumably through one or sev-eral of the following channel types: mechanosensitivechannels (MSC), store-operated channels (SOC) orsecond messenger operated channels (SMOC). Vesi-cle fusion with the plasma membrane is triggered withinseconds to minutes. In both cell types, intracellularstores also contribute to the increase in cytoplasmicCa2+ concentration (not shown).

Stretch and ATP - induced Surfactant Exocytosis Cell Physiol Biochem 2010;25:01-12

4

(ECM)/integrin signalling, gene expression, cytokine re-lease and type II cell function/phenotype has been re-viewed in detail elsewhere [22, 27, 54-57]. Hinderingintegrin interaction with the ECM by RGD (Arg-Gly-Asp)peptides inhibits the stretch-induced Ca2+ signal (G. Fois,unpublished observation). Recently, it was shown thatvariable stretch patterns influence surfactant secretion[58]. These observations leave space for speculationsthat mechanically induced signalling molecules might af-fect LB exocytosis, however possible links between fo-cal adhesions and Ca2+ permeable channels are yet un-clear.

Possible causes of ATP release in thealveolus

As noted, stretch during a deep lung inflation is con-sidered the most important if not the only physiologicallyrelevant stimulus of surfactant secretion [23-25, 27, 59-64], and stretch-induced cellular ATP release could rep-resent a key element of pulmonary alveolarmechanotransduction. An autocrine mechanism ofpurinergic (ATP) stimulation was proposed after hypot-onic swelling in a surrogate cell line of type II pneumocytes(A549 cells) [65], but it is still unclear if osmotic cell vol-ume changes occur in alveolar cells in vivo at all. Theintriguing question whether or not ATP is indeed involvedin stretch-induced surfactant secretion in the native lungremains unclear. In 1983, Gilfillan et al. [66] presentedfirst evidence for extracellular ATP as a stimulator ofsurfactant secretion using a perfused rat lung slice prepa-ration. Their work was inspired by the discovery ofpurinergic nerves [67], which utilize ATP as aneuro(co)transmitter and innervate several visceral or-gans including the lung, terminating at sites close to al-veolar type II cells [68]. Although a clear physiologicalrole of ATP release from pulmonary purinergic nerveshas never been established, ATP emerged as the mostpotent physiological agonist of surfactant secretion, de-spite its mysterious origin and presence under variousconditions of stimulation. One possible route of ATP re-lease is through anion channels (reviewed in detail [69]).However, although various anion channels have been iden-tified in fetal and adult pneumocytes [70], their involve-ment in alveolar ATP release has not yet been demon-strated. It is unlikely that membrane stress alone is thereason for ATP release from cells under physiologicalconditions, because moderate amounts of stretch are ingeneral tolerated without signs of membrane damage [35,

36, 71, 72]. However, ATP may exit all cells under condi-tions of cell damage, and this could be the case in hyper-inflation-induced lung injury. Indeed, Gajic et al. foundsigns of reversible cell membrane stress failure in ratsventilated with high tidal volumes using propidium iodideas a cell-impermeable marker of cell damage [72]. Inaddition, we found ultrastructural evidence for membranedamage in type II pneumocytes stretched on silastic mem-branes [71]. In summary, in pathophysiological conditions,ATP leakage from wounded cells could influence type IIcells and surfactant secretion in an autocrine/paracrineway.

Interestingly, new findings suggest that purinergic(ATP) control of surfactant secretion could be stimulatedby bacterial lipopolysaccharides (LPS), presumably viaupregulation of purinergic receptors of the P2Y2 subtype(see below), resulting in an increased Ca2+ signal and apronounced exocytotic response of LBs to treatment withATP [73]. Hence, sensitation of the purinergic systemduring inflammation might augment physiological stimuli.

Purinergic receptors and their signallingcascades in type II pneumocytes

Since ATP is a very potent stimulus of surfactantsecretion [74-77], the purinergic signalling pathway hasbeen intensively investigated and is detailed in severalexcellent reviews [1, 17, 18, 21, 78-80]. ATP and UTPare equally potent to stimulate surfactant secretion [81],indicating signalling via a P2Y2 receptor (formally termedP2U receptor), consistent with pharmacological data [74].In addition, surfactant secretion is also stimulated by ad-enosine [75], and all its receptor subtypes (A1, A2A, A2Band A3) have been identified in isolated type II cells [82].

The ATP-induced Ca2+ signal in type II cells con-sists of at least 2 phases, the “peak” and the “plateau”phase, analogous to a multitude of other cell types [83].The “peak” is independent of extracellular Ca2+ and hencea result of intracellular Ca2+ release from stores, pre-sumably the endoplasmic reticulum [83]. The “plateau”depends on extracellular Ca2+ and is most likely a resultof Ca2+ entry through the plasma membrane [83]. The“plateau” is not as stable as in many other cell types butfrequently superimposed by transient Ca2+ elevations ofyet unknown origin that coincide with single LB fusionswith the plasma membrane [84]. Ion channels which areresponsible for the ATP-induced Ca2+ influx and “pla-teau” phase in type II pneumocytes are yet unknown,nor is their mechanism of activation. In analogy to stretch-


5

induced Ca2+ entry (see above), SOCs and/or SMOCsmay account for ATP-induced Ca2+ entry (Fig. 1). Sincetype II cells are subject to considerable changes of shape(and possibly cell volume) after stimulation with ATP (un-published observation), not even MSCs can be excludedin this context.

Type II cell stimulation with ATP is coupled to bothadenylate cyclase (AC; cAMP/PKA) and phospholipaseC (PLC; IP3/DAG/PKC) signalling [85, 86]. cAMP isalso generated due to stimulation of ß2-adrenergicreceptors with isoproterenol [87], which may be impor-tant during labor [88]. Accordingly, cell-permeable cAMPanalogues themselves stimulate surfactant secretion [89].The mechanism by which cAMP elicits this exocytoticresponse is not yet known, but its potency is much lowerthan that of an elevation of the cytoplasmic Ca2+ concen-tration or phorbol esthers (Haller and Dietl, unpublishedobservation), which also stimulate secretion (see below).

Activation of PLC results in phosphoinositide (PIP2)breakdown and generation of inositol-1,4,5-trisphosphate(IP3) and diacylglycerol (DAG) [74]. The PLC isoformPLC-β3 was identified by RT-PCR and immunoblottingas the only isoform present in type II cells [82]. DAGgeneration in type II cells in response to extracellular ATPis biphasic: The first peak occurs immediately (about 10s) after ATP treatment and coincides with IP3 formationas a result of PLC activation [74]. The second DAGpeak follows with a delay of 10 - 15 min and coincideswith formation of phosphatidic acid (PA) or - in the pres-ence of ethanol - phosphatidylethanol, indicating thecoactivation of a phosphatidylcholine-specific phospholi-pase D (PLD) [74, 81, 90]. mRNA for both PLDisoforms, PLD1 and PLD2, have been identified by RT-PCR in type II cells [80].

ATP-induced DAG activates protein kinase C(PKC), and again, several PKC-isoforms have been iden-tified by RT-PCR in type II cells [82, 91, 92]. DAG, re-leased from a caged compound by UV-photolysis, or cell-permeable phorbol esthers (like TPA, OAG) that directlyactivate PKC, are themselves strong stimulators of sur-factant secretion [83, 93], further supporting that PKC isinvolved in downstream stimulation of surfactant secre-tion via extracellular ATP. In fact, phorbol esthersdegranulate type II cells quite effectively without a con-comitant elevation of the cytoplasmic Ca2+ concentra-tion, but intracellular Ca2+ chelation abolishes this effect[83]. It was therefore proposed that PKC sensitizes theexocytotic machinery for the action of Ca2+ [83, 94], pos-sibly by phosphorylation of SNAP-25 as demonstrated inan insulin-secreting cell line [95].

In this context it is reasonable to assume that IP3formation accounts for ATP-induced Ca2+ mobilizationfrom intracellular stores [96, 97]. However, the effectsof IP3 were never directly investigated in type IIpneumocytes. It is not clear if Ca2+ stores mobilized byATP are different from or overlap with Ca2+ stores mobi-lized by stretch.

The process of surfactant exocytosis

The described orchestrated activity of bioactive com-pounds generated by stretch or purinergic stimulationmakes a single mechanism or molecular target ofexocytosis quite unlikely. Nevertheless, the elevation of[Ca2+]c appears to be a central common component bywhich LB exocytosis from alveolar type II pneumocytesis triggered. This assumption is based on the followingfindings:

1. A variety of Ca2+-dependent agonists and Ca2+

ionophores stimulates surfactant secretion, most specifi-cally flash photolysis of caged Ca2+ [83, 98-100].

2. Intracellular Ca2+ chelation blocks LB exocytosisstimulated by different agonists [83, 100].

3. There is an almost linear correlation between theintegrated [Ca2+]c over time and the amount of surfactantsecretion after stimulation with ATP [83].

Since exocytosis is an extremely conserved mecha-nism in evolution, the predominant role of Ca2+ in this celltype is not surprising. However, in contrast to synaptic orneuroendocrine secretion, where secretory vesicle fusionoccurs within < one millisecond up to > hundred millisec-onds following the rise of [Ca2+]c [101, 102], the kineticsand time scale of surfactant release are delayed by sev-eral orders of magnitude: The delay between the ATP-induced Ca2+ signal and LB fusion with the plasma mem-brane is between seconds and several minutes, and thedelay between LB fusion and surfactant release is of thesame order of magnitude, occasionally even longer (hours)[84, 96, 99]. The reasons for this discrepancy betweentype II cell and neuroendocrine secretion are probablymanifold (see also Fig. 1), but mostly related to structuralfeatures: First, there is neither morphological nor func-tional evidence in favour of so-called “docked” vesiclesin type II cells. It rather appears that a few LBs arebulging out the plasma membrane, resulting in tongue-like folds of the plasma membrane after fusion [10]. Sec-ond, as noted above, epithelial cells lack voltage-gatedCa2+ channels and thus have no “active zones”, as presentin presynaptic terminals.


6

As summarised in Fig. 2, the exocytotic process ofsurfactant could be divided into three distinct steps, whichare differently controlled. Each one can be rate-limitingfor the secretion of surfactant:

1. The prefusion phase: This denotes all events thatmake the LB fusion-competent, including trafficking anddocking of the LB to the plasma membrane, creating amorphological and biochemical contiguity that enables li-pid merger and fusion pore formation.

2. The hemifusion phase and fusion: The exocytotichemifusion state (“fusion-through-hemifusion” theory) isa postulate (reviewed in detail by [103]) which denotesthe merger of the outer leaflet of the limiting vesicle mem-brane with the inner leaflet of the plasma membrane.Experimental evidence for hemifusion is scarce and indi-rect [104, 105], however our own observations by darkfieldmicroscopy provide evidence in favour of hemifusion in-termediates in the type II cell, with a duration from notmeasurable up to about 9s [106].

Fusion pore formation takes place when the vesiclemembrane and plasma membrane become a lipid bilayercontinuum, and thus the vesicle lumen and the extracel-lular space an aqueous continuum. These steps have beenintensively investigated in biological and artificial systemsand are hallmarks of exocytosis [107].

3. The postfusion phase: This denotes fusion poreexpansion and release of LB contents. In the type II cell,fusion of a LB with the plasma membrane (synonymouswith fusion pore formation) can precede surfactant re-

lease (secretion) for long periods (up to hours under cellculture conditions, see also below).

Control of the prefusion phase

LB fusion with the plasma membrane is a stereotypicresponse to an elevation of [Ca2+]c, triggered eg. by UVflash photolysis of caged Ca2+ [99], the Ca2+ ionophoreionomycin [100, 108-110], cell stretch [35, 36], or purinergicstimulation (as discussed above). The threshold [Ca2+]cfor LB fusion is about 320 nM and thus far lower than incells with small vesicles (up to hundreds of µM) [99]. Incontrast to far more intensively investigated neurotrans-mitter release [107], the molecular mechanisms by whichCa2+ elicits LB fusion are still less clear. SNARE (solu-ble N-ethylmaleimide-sensitive-factor attachmentreceptor) proteins and the presumed Ca2+ sensorsynaptotagmin are major candidates for LB fusion , andsynaptotagmin II knockout mice have indeed impairedstimulated mucin secretion from airway epithelial cells,which shares many features with surfactant secretion[112, 113]. Yet, no information is available on surfactantsecretion in these mice.

Another discussed Ca2+ sensor that might be respon-sible for LB fusion is synexin (annexin VII), a GTPhydrolyzing and phospholipid binding protein, displayingfusogenic activity in various cell types [114]. Several stud-ies suggest involvement of synexin in Ca2+-dependent LB

Fig. 2. Top: Schematic illustration of the threephases of surfactant secretion from type IIpneumocytes, indicated by different back-ground colors. Prefusion, hemifusion andpostfusion. The hemifusion and postfusionphases are separated by the instance of fusionpore formation (fusion). Bottom: Original trac-ing of the darkfield light intensity measuredfrom a single LB within a living type II cell.Arrows indicate that phase 1 of the darkfieldscattered light intensity decrease (SLID) cor-responds to the presumed hemifusion phase,and phase 2 to the postfusion phase. Modifiedfrom Ref [106]. For details see text.


7

fusion with the plasma membrane [115-119]. This isconsistent with the finding that the non-hydrolyzeable GTPanalogue GTPγS stimulates LB fusion with the plasmamembrane without stimulating LB-LB fusion [120].

Another way by which Ca2+ could mediateLB fusion with the plasma membrane is the disassemblyof cortical actin, which is located beneath the plasmamembrane. The idea that exocytotic fusion is limitedby the cortical F-actin network, which represents a physi-cal barrier, preventing vesicle contact with the plasmamembrane, was proposed as a general principle forexocytosis in non-excitable cells [121]. The notion thatexocytotic fusion is enabled whenever the fusion hin-drance by cortical actin is removed gains support byrecent fusion studies on proteoliposomes and synaptic vesi-cles, showing that the SNARE complex syntaxin 1,SNAP-25, and synaptobrevin can act as a constitutivelyactive fusion machine, independently of synaptotagminand Ca2+ [122]. Evidence that disassembly of the actinnetwork does in fact enhance exocytotic LB fusion comesfrom a study where basal surfactant secretion wasaugmented after decreasing cellular F-actin levels withbotulinum C2 toxin [123]. Beside being a fusion clampthat inhibits fusion, actin may promote exocytosis byvarious mechanisms at a pre- or postfusion stage(see below). Possible roles for actin in the exocytoticprocess have recently been summarized in an excellentreview by Malacombe et al. [124]. A possible Ca2+ sen-sor for this task is scinderin, a Ca2+-dependent actin fila-ment-severing protein, which binds phosphatidylserine,PIP2 and actin [124]. A role for scinderin was demon-strated in mucous cells [125], but information in type IIcells is yet lacking.

Control of the hemifusion phase

As a general rule, hemifusion is favored by lipidsthat support negative curvature of the membraneleaflets (facilitating stalk formation), such as fatty acids,whereas fusion pore formation and expansion is favoredby positive curvature lipids, such as lysophospholipids[126]. It was proposed that vesicles transiently hemifusedwith the presynaptic membrane enable the extremely fastkinetics of neurotransmitter release (< 100 µs after therise of [Ca2+]c) [127], and this is consistent with EM andFRAP investigations revealing docked vesicles in ahemifused state [104, 105]. Owing to the long delay(several seconds) between the elevation of [Ca2+]c andfusion pore formation in LBs [99], it is very unlikely

that LBs reside in a “docked”, hemifused state.Nevertheless, using a newly developed hemifusion assaybased on darkfield scattered light intensity decrease(Fig. 2), we have recently shown that hemifusion inter-mediates in type II pneumocytes can last for up to about9 seconds [106]. It is yet unclear if LB hemifusion inter-mediates are always destined to proceed to fusion, orif they can be transient and revert to a prefused state.In any case they represent a significant stage ofsurfactant exocytosis that is potentially subject toregulation by SNARE proteins or lipid metabolites.

Among the discussed bioactive lipid metabolitesgenerated by purinergic stimulation that might promoteLB fusion, PA is a particularly promising candidatebecause it induces negative plasma membrane curvaturedue to its small polar head group in combination withtwo fatty-acyl side chains [128], possibly promotinglipid merger with the vesicle membrane and hemi-fusion[129]. Although direct evidence for this mechanism hasnever been presented in type II cells, it is supported bythe observation that some type II cells exhibit a biphasicfusion activity (immediately after ATP treatment and> 10 min later), where the early response coincides withCa2+ mobilization, whereas the delayed one occurs witha delay reported for PA generation [84].

Mechanisms to control surfactant releasein the postfusion phase

Our knowledge about the release mechanism ofsurfactant from native type II cells in the lung still mainlycomes from electron microscopy (EM) investigations,where tubular myelin, a highly ordered array ofmembranes found by transmission EM in pulmonaryalveoli, was postulated as the extracellular conversionproduct of released LBs [130, 131]. Some imagesdisplay fusion pores of various diameters (up to > 1 µm),through which surfactant appears to be squeezed(example in Ref. [80]). However, a dynamic resolutionof this process within the intact lung is still beyondthe limits of current imaging techniques.

Nevertheless, advanced microscopy techniquesdo allow the visualization of the entire exocytotic process– including fusion pore formation, fusion pore expansionand surfactant release in either primary cultured type IIpneumocytes [84, 96, 132, 133] or in situ (isolatedperfused lung), however yet with limited resolution[37]. Using fluorescence microscopy in isolated type IIcells, tubular myelin cannot be clearly identified, and it


8

appears that the hydrophobic, poorly soluble complex ofsurfactant must be actively squeezed through areluctantly opening fusion pore. Several experimental dataare in line with this notion:

1. Fusion pores in type II cells have the tendencyto expand slowly [84]. The reason for this slowexpansion is not yet clear but may well be related tocytoskeletal elements. An elevation of [Ca2+]c acceler-ates pore expansion [84].

2. Fusion pores in type II cells can act asmechanical barriers for release. This was concludedfrom experiments using laser tweezers as force genera-tors [134].

3. An actin coat must be formed around the fusedLB before surfactant can be squeezed out, probablyby contraction of the actin coat [133]. These observa-tions using GFP-actin-transfected pneumocytes wereconsistent with earlier observations that actin re-organization accompanies LB exocytosis [135, 136].

Prior to LB contraction by the actin coat, thefused LB transiently swells, presumably by fluid uptakethrough the fusion pore [106]. The physiological signifi-cance of postfusion LB swelling is yet unknown, althoughthis may represent a mechanical load to the limiting LBmembrane and surrounding structures. Postfusion vesi-cle swelling was observed in numerous secretory celltypes [137]. Swelling of the vesicle and hydration of vesi-cle matrix might be important for fusion pore stabilizationand expansion in mast cells [138].

Intracellular Ca2+ immobilization with the chelatorBAPTA-AM, as well as the PLD inhibitor C2-ceramidehindered ATP-induced actin coat formation in type IIcells [133]. In the absence of an actin coat, however,cells were unable to secrete surfactant, and fusedLBs remained in this position (“wait” position) as filledbags connected with the plasma membrane [133].This suggests that Ca2+ and PLD activation (in responseto ATP) may control the postfusion (release) phase ofsurfactant exocytosis.

Is actin-powered release the reason for fastsurfactant secretion in vivo?

There is ample evidence that surfactant secretionby pharmacological stimulation or stretch in vivo is fastand occurs within min (reviewed in detail in [21, 139]).Isolated type II pneumocytes in vitro, however, exhibit a

considerable variability in the time course ofsurfactant release from single LBs, ranging fromminutes to hours [84]. Recent experiments usingGFP-actin-transfected pneumocytes suggest that a slowtime course of release relates with the inability to pro-duce an actin coat around fused LBs [133]. Hence, itmay be that the fast surfactant secretion observed invivo is due to a more effective actin coating as com-pared to isolated cells.

Outlook

The slow process of stimulated surfactant exocytosisrenders the type II pneumocyte an ideal cell model toinvestigate time-resolved actions of physiologicallyrelevant modulators of exocytosis.

Currently, one of the most intriguing questions is howactin coat formation is limited to the fused LB withoutaffecting all LBs in a cell stimulated with ATP. It appearsthat there are spacially and/or temporarily confinedprocesses that cannot spread throughout the cell. Yu andBement recently disclosed a signalling pathway in oocytesinvolving a PLD-dependent DAG incorporation into thegranule membrane after fusion, triggering Cdc42/N-WASP-induced actin assembly [140]. Although this stillhas to be investigated in type II cells, PLD would beexpected to primarily control the post-fusion phase,because neither alcohol, which inhibits PLD-inducedDAG generation, nor the PLD inhibitor C2 ceramide,blocked LB fusion activity [83, 133]. As outlined above,a potential way by which lipid metabolites might controlsurfactant exocytosis is the promotion of hemifusion aswell as the expansion of the fusion pore.

Inducible, type II pneumocyte-specific knock-out animals of the P2Y2 receptor would probably be ofgreat help to distinguish stretch- from ATP-dependentsignalling pathways and to clarify potential autocrine(ATP release) mechanisms involved in stretch-inducedsurfactant secretion.

Acknowledgements

This work was supported by the DeutscheForschungsgemeinschaft, grant D1402 (to P.D.).B.L. is supported by the Alfried Krupp Prize of theKrupp Foundation.


9

References

1 Rooney SA, Young SL, Mendelson CR:Molecular and cellular processing of lungsurfactant. FASEB J 1994;8:957-967.

2 van Golde LM, Batenburg JJ, RobertsonB: The pulmonary surfactant system:biochemical aspects and functional sig-nificance. Physiol Rev 1988;68:374-455.

3 Von Neergaard K: Neue Auffassungenüber einen Grundbegriff derAtemmechanik. Z Ges Exptl Med1929;66:373-394.

4 Clements JA: Lung surfactant: a personalperspective. Annu Rev Physiol1997;59:1-21.

5 Pattle RE: Surface lining of lung alveoli.Physiol Rev 1965;45:48-79.

6 Avery ME, Mead J: Surface properties inrelation to atelectasis and hyaline mem-brane disease. Am J Dis Child1959;97:517-523.

7 Campiche M: Les inclusions lamellairesdes cellules alveolaires dans le pneumondu raton. J Ultrastruct Res 1960;3:302-312.

8 Perez-Gil J: Structure of pulmonary sur-factant membranes and films: The roleof proteins and lipid-protein interactions.Biochim Biophys Acta 2008;1778:1676-1695.

9 Crapo JD, Barry BE, Gehr P, BachofenM, Weibel ER: Cell number and cell char-acteristics of the normal human lung. AmRev Respir Dis 1982;126:332-337.

10 Ryan US, Ryan JW, Smith DS: Alveolartype II cells: studies on the mode of re-lease of lamellar bodies. Tissue Cell1975;7:587-599.

11 Kikkawa Y, Smith F: Cellular and bio-chemical aspects of pulmonary sur-factant in health and disease. Lab Invest1983;49:122-139.

12 Veldhuizen R, Nag K, Orgeig S, PossmayerF: The role of lipids in pulmonary sur-factant. Biochim Biophys Acta1998;1408:90-108.

13 Askin FB, Kuhn C: The cellular origin ofpulmonary surfactant. Lab Invest1971;25:260-268.

14 Veldhuizen EJ, Haagsman HP: Role ofpulmonary surfactant components insurface film formation and dynamics.Biochim Biophys Acta 2000;1467:255-270.

15 Schurch S, Green FH, Bachofen H: For-mation and structure of surface films:captive bubble surfactometry. BiochimBiophys Acta 1998;1408:180-202.

16 Bates SR, Tao JQ, Notarfrancesco K,DeBolt K, Shuman H, Fisher AB: Effectof Surfactant Protein-A on GranularPneumocyte Surfactant Secretion inVitro. Am J Physiol Lung Cell MolPhysiol 2003;285:L1055-65.

17 Andreeva AV, Kutuzov MA, Voyno-Yasenetskaya TA: Regulation of sur-factant secretion in alveolar type II cells.Am J Physiol Lung Cell Mol Physiol2007;293:L259-L271.

18 Chander A, Fisher AB: Regulation of lungsurfactant secretion. Am J Physiol1990;258:L241-L253.

19 Dietl P, Haller T: Exocytosis of lung sur-factant: from the secretory vesicle tothe air-liquid interface. Annu Rev Physiol2005;67:595-621.

20 Mason RJ: Surfactant secretion; inRobertson B, van Golde LM, BatenburgJJ (eds): Pulmonary Surfactant: frommolecular biology to clinical practice.Amsterdam, Elsevier Science Publishers,1992, pp 295-312.

21 Wright JR, Dobbs LG: Regulation of pul-monary surfactant secretion and clear-ance. Annu Rev Physiol 1991;53:395-414.

22 Dobbs LG, Gutierrez JA: Mechanicalforces modulate alveolar epithelialphenotypic expression. Comp BiochemPhysiol A Mol Integr Physiol2001;129:261-266.

23 Massaro GD, Massaro D: Morphologicevidence that large inflations of the lungstimulate secretion of surfactant. Am RevRespir Dis 1983;127:235-236.

24 Nicholas TE, Barr HA: Control of re-lease of surfactant phospholipids in theisolated perfused rat lung. J Appl Physiol1981;51:90-98.

25 Nicholas TE, Power JH, Barr HA: Thepulmonary consequences of a deepbreath. Respir Physiol 1982;49:315-324.

26 Oyarzun MJ, Clements JA: Control oflung surfactant by ventilation, adrener-gic mediators, and prostaglandins in therabbit. Am Rev Respir Dis 1978;117:879-891.

27 Wirtz HR, Dobbs LG: The effects ofmechanical forces on lung functions.Respir Physiol 2000;119:1-17.

28 Verbrugge SJ, Lachmann B, Kesecioglu J:Lung protective ventilatory strategies inacute lung injury and acute respiratorydistress syndrome: from experimentalfindings to clinical application. ClinPhysiol Funct Imaging 2007;27:67-90.

29 Azeloglu EU, Bhattacharya J, Costa KD:Atomic force microscope elastographyreveals phenotypic differences in alveo-lar cell stiffness. J Appl Physiol2008;105:652-661.

30 Gefen A, Elad D, Shiner RJ: Analysis ofstress distribution in the alveolar septaof normal and simulated emphysematiclungs. J Biomech 1999;32:891-897.

31 Gefen A, Halpern P, Shiner RJ, SchroterRC, Elad D: Analysis of mechanicalstresses within the alveolar septa leadingto pulmonary edema. Technol HealthCare 2001;9:257-267.

32 Gil J, Weibel ER: Morphological study ofpressure-volume hysteresis in rat lungsfixed by vascular perfusion. RespirPhysiol 1972;15:190-213.

33 Tschumperlin DJ, Margulies SS: Alveolarepithelial surface area-volume relation-ship in isolated rat lungs. J Appl Physiol1999;86:2026-2033.

34 Gerstmair A, Fois G, Innerbichler S, DietlP, Felder E: A device for simultaneouslive cell imaging during uni-axial mechani-cal strain or compression. J Appl Physiol2009;107:613-620.

35 Frick M, Bertocchi C, Jennings P, HallerT, Mair N, Singer W, Pfaller W, Ritsch-Marte M, Dietl P: Ca2+ entry is essentialfor cell strain-induced lamellar body fu-sion in isolated rat type II pneumocytes.Am J Physiol Lung Cell Mol Physiol2004;286:L210-L220.

36 Wirtz HR, Dobbs LG: Calcium mobiliza-tion and exocytosis after one mechani-cal stretch of lung epithelial cells. Sci-ence 1990;250:1266-1269.

37 Ashino Y, Ying X, Dobbs LG,Bhattacharya J: [Ca2+]i oscillations regu-late type II cell exocytosis in the pulmo-nary alveolus. Am J Physiol Lung CellMol Physiol 2000;279:L5-13.

38 Boitano S, Safdar Z, Welsh DG,Bhattacharya J, Koval M: Cell-cell in-teractions in regulating lung function. AmJ Physiol Lung Cell Mol Physiol2004;287:L455-L459.

39 Patel AS, Reigada D, Mitchell CH, BatesSR, Margulies SS, Koval M: Paracrinestimulation of surfactant secretion byextracellular ATP in response to me-chanical deformation. Am J Physiol LungCell Mol Physiol 2005;289:L489-L496.

40 Frick M, Siber G, Haller T, Mair N, DietlP: Inhibition of ATP-induced surfactantexocytosis by dihydropyridine (DHP)derivatives: a non-stereospecific,photoactivated effect and independentof L-type Ca2+ channels. BiochemPharmacol 2001;61:1161-1167.

41 Mair N, Frick M, Bertocchi C, Haller T,Amberger A, Weiss H, Margreiter R, StreifW, Dietl P: Inhibition by cytoplasmicnucleotides of a new cation channel infreshly isolated human and rat type IIpneumocytes. Am J Physiol Lung CellMol Physiol 2004;287:L1284-L1292.

42 Drummond HA, Grifoni SC, Jernigan NL:A new trick for an old dogma: ENaC pro-teins as mechanotransducers in vascularsmooth muscle. Physiology (Bethesda)2008;23:23-31.


10

43 Hamill OP: Twenty odd years of stretch-sensitive channels. Pflugers Arch2006;453:333-351.

44 Birder LA, Nakamura Y, Kiss S, NealenML, Barrick S, Kanai AJ, Wang E, RuizG, De Groat WC, Apodaca G, Watkins S,Caterina MJ: Altered urinary bladder func-tion in mice lacking the vanilloid receptorTRPV1. Nat Neurosci 2002;5:856-860.

45 O’Neil RG, Heller S: Themechanosensitive nature of TRPV chan-nels. Pflugers Arch 2005;451:193-203.

46 Clapham DE: TRP channels as cellularsensors. Nature 2003;426:517-524.

47 Maroto R, Raso A, Wood TG, KuroskyA, Martinac B, Hamill OP: TRPC1 formsthe stretch-activated cation channel invertebrate cells. Nat Cell Biol2005;7:179-185.

48 Gottlieb P, Folgering J, Maroto R, RasoA, Wood TG, Kurosky A, Bowman C,Bichet D, Patel A, Sachs F, Martinac B,Hamill OP, Honore E: Revisiting TRPC1and TRPC6 mechanosensitivity.Pflugers Arch 2008;455:1097-1103.

49 Christensen AP, Corey DP: TRP chan-nels in mechanosensation: direct or indi-rect activation? Nat Rev Neurosci2007;8:510-521.

50 Kunert-Keil C, Bisping F, Kruger J,Brinkmeier H: Tissue-specific expressionof TRP channel genes in the mouse andits variation in three different mousestrains. BMC Genomics 2006;7:159.

51 Thastrup O, Dawson AP, Scharff O, FoderB, Cullen PJ, Drobak BK, Bjerrum PJ,Christensen SB, Hanley MR:Thapsigargin, a novel molecular probefor studying intracellular calcium releaseand storage. Agents Actions 1989;27:17-23.

52 Hoth M, Penner R: Depletion of intrac-ellular calcium stores activates a calciumcurrent in mast cells. Nature1992;355:353-356.

53 Cahalan MD: STIMulating store-oper-ated Ca2+ entry. Nat Cell Biol2009;11:669-677.

54 Garcia CS, Prota LF, Morales MM,Romero PV, Zin WA, Rocco PR: Under-standing the mechanisms of lung me-chanical stress. Braz J Med Biol Res2006;39:697-706.

55 Ingber DE: Mechanosensation throughintegrins: cells act locally but think glo-bally. Proc Natl Acad Sci U S A2003;100:1472-1474.

56 Orr AW, Helmke BP, Blackman BR,Schwartz MA: Mechanisms ofmechanotransduction. Dev Cell2006;10:11-20.

57 Waters CM, Sporn PH, Liu M, FredbergJJ: Cellular biomechanics in the lung. AmJ Physiol Lung Cell Mol Physiol2002;283:L503-L509.

58 Arold SP, Bartolak-Suki E, Suki B: Vari-able stretch pattern enhances surfactantsecretion in alveolar type II cells in cul-ture. Am J Physiol Lung Cell Mol Physiol2009;296:L574-L581.

59 Dietl P, Frick M, Mair N, Bertocchi C,Haller T: Pulmonary consequences of adeep breath revisited. Biol Neonate2004;85:299-304.

60 Faridy EE: Effect of distension on re-lease of surfactant in excised dogs’ lungs.Respir Physiol 1976;27:99-114.

61 Hildebran JN, Goerke J, Clements JA:Surfactant release in excised rat lung isstimulated by air inflation. J Appl Physiol1981;51:905-910.

62 McClenahan JB, Urtnowski A: Effect ofventilation on surfactant, and its turno-ver rate. J Appl Physiol 1967;23:215-220.

63 Nicholas TE, Power JH, Barr HA: Sur-factant homeostasis in the rat lung dur-ing swimming exercise. J Appl Physiol1982;53:1521-1528.

64 Nicholas TE, Barr HA: The release ofsurfactant in rat lung by brief periods ofhyperventilation. Respir Physiol1983;52:69-83.

65 Tatur S, Groulx N, Orlov SN, GrygorczykR: Ca2+-dependent ATP release from A549cells involves synergistic autocrine stimu-lation by coreleased uridine nucleotides.J Physiol 2007;584:419-435.

66 Gilfillan AM, Hollingsworth M, JonesAW: The pharmacological modulationof [3H]-disaturated phosphatidylcholineoverflow from perifused lung slices ofadult rats: a new method for the study oflung surfactant secretion. Br J Pharmacol1983;79:363-371.

67 Burnstock G: Purinergic nerves.Pharmacol Rev 1972;24:509-581.

68 Welsch U, Muller W: [Electron micro-scopic studies of reptilian lung innerva-tion]. Z Mikrosk Anat Forsch1980;94:435-444.

69 Sabirov RZ, Okada Y: ATP release viaanion channels. Purinergic Signal2005;1:311-328.

70 O’Grady SM, Lee SY: Chloride and po-tassium channel function in alveolar epi-thelial cells. Am J Physiol Lung Cell MolPhysiol 2003;284:L689-L700.

71 Felder E, Siebenbrunner M, Busch T, FoisG, Miklavc P, Walther P, Dietl P: Me-chanical strain of alveolar type II cellsin culture: changes in the transcellularcytokeratin network and adaptations.Am J Physiol Lung Cell Mol Physiol2008;295:L849-L857.

72 Gajic O, Lee J, Doerr CH, Berrios JC,Myers JL, Hubmayr RD: Ventilator-in-duced cell wounding and repair in the in-tact lung. Am J Respir Crit Care Med2003;167:1057-1063.

73 Garcia-Verdugo I, Ravasio A, Garcia dePE, Synguelakis M, Ivanova N,Kanellopoulos J, Haller T: Long termexposure to LPS enhances the rate ofstimulated exocytosis and surfactant se-cretion in alveolar type II cells andupregulates P2Y2 receptor expression.Am J Physiol Lung Cell Mol Physiol2008;295(4):L708-17.

74 Griese M, Gobran LI, Rooney SA: ATP-stimulated inositol phospholipid metabo-lism and surfactant secretion in rat typeII pneumocytes. Am J Physiol1991;260:L586-L593.

75 Gilfillan AM, Rooney SA: Purinoceptoragonists stimulate phosphatidylcholinesecretion in primary cultures of adult rattype II pneumocytes. Biochim BiophysActa 1987;917:18-23.

76 Dorn CC, Rice WR, Singleton FM: Cal-cium mobilization and response recov-ery following P2-purinoceptor stimula-tion of rat isolated alveolar type II cells.Br J Pharmacol 1989;97:163-170.

77 Rice WR: Effects of extracellular ATPon surfactant secretion. Ann N Y AcadSci 1990;603:64-74.

78 Mason RJ, Voelker DR: Regulatorymechanisms of surfactant secretion.Biochim Biophys Acta 1998;1408:226-240.

79 Rooney SA: Regulation of surfactant se-cretion (book); in Rooney SA (ed): LungSurfactant: Cellular and MolecularProcessing. Austin, Texas, R.G.LandesCompany, 1998, pp 139-161.

80 Rooney SA: Regulation of surfactant se-cretion. Comp Biochem Physiol A MolIntegr Physiol 2001;129:233-243.

81 Gobran LI, Xu ZX, Lu Z, Rooney SA:P2u purinoceptor stimulation of sur-factant secretion coupled tophosphatidylcholine hydrolysis in typeII cells. Am J Physiol 1994;267:L625-L633.

82 Gobran LI, Xu ZX, Rooney SA: PKCisoforms and other signaling proteinsinvolved in surfactant secretion in de-veloping rat type II cells. Am J Physiol1998;274:L901-L907.

83 Frick M, Eschertzhuber S, Haller T, MairN, Dietl P: Secretion in alveolar type IIcells at the interface of constitutive andregulated exocytosis. Am J Respir CellMol Biol 2001;25:306-315.

84 Haller T, Dietl P, Pfaller K, Frick M,Mair N, Paulmichl M, Hess MW, Furst J,Maly K: Fusion pore expansion is a slow,discontinuous, and Ca2+-dependent proc-ess regulating secretion from alveolartype II cells. J Cell Biol 2001;155:279-289.

85 Gobran LI, Rooney SA: Adenylate cy-clase-coupled ATP receptor and surfactantsecretion in type II pneumocytes fromnewborn rats. Am J Physiol1997;272:L187-L196.


11

86 Warburton D, Buckley S, Cosico L: P1and P2 purinergic receptor signal trans-duction in rat type II pneumocytes. JAppl Physiol 1989;66:901-905.

87 Brown LA, Longmore WJ: Adrenergicand cholinergic regulation of lung sur-factant secretion in the isolated perfusedrat lung and in the alveolar type II cell inculture. J Biol Chem 1981;256:66-72.

88 Marino PA, Rooney SA: The effect oflabor on surfactant secretion in newbornrabbit lung slices. Biochim Biophys Acta1981;664:389-396.

89 Chander A: Regulation of lung surfactantsecretion by intracellular pH. Am JPhysiol 1989;257:L354-L360.

90 Rooney SA, Gobran LI: Activation ofphospholipase D in rat type IIpneumocytes by ATP and other sur-factant secretagogues. Am J Physiol1993;264:L133-L140.

91 Chander A, Sen N, Wu AM, Spitzer AR:Protein kinase C in ATP regulation oflung surfactant secretion in type II cells.Am J Physiol 1995;268:L108-L116.

92 Gobran LI, Rooney SA: Surfactant secre-tagogue activation of protein kinase Cisoforms in cultured rat type II cells. AmJ Physiol 1999;277:L251-L256.

93 Sano K, Voelker DR, Mason RJ: Involve-ment of protein kinase C in pulmonarysurfactant secretion from alveolar typeII cells. J Biol Chem 1985;260:12725-12729.

94 Hille B, Billiard J, Babcock DF, NguyenT, Koh DS: Stimulation of exocytosiswithout a calcium signal. J Physiol1999;520 Pt 1:23-31.

95 Shu Y, Liu X, Yang Y, Takahashi M, GillisKD: Phosphorylation of SNAP-25 atSer187 mediates enhancement ofexocytosis by a phorbol ester in INS-1cells. J Neurosci 2008;28:21-30.

96 Haller T, Ortmayr J, Friedrich F, VolklH, Dietl P: Dynamics of surfactant re-lease in alveolar type II cells. Proc NatlAcad Sci U S A 1998;95:1579-1584.

97 Rice WR, Singleton FM: P2Y-purinoceptor regulation of surfactantsecretion from rat isolated alveolar typeII cells is associated with mobilization ofintracellular calcium. Br J Pharmacol1987;91:833-838.

98 Dobbs LG, Gonzalez RF, Marinari LA,Mescher EJ, Hawgood S: The role of cal-cium in the secretion of surfactant by ratalveolar type II cells. Biochim BiophysActa 1986;877:305-313.

99 Haller T, Auktor K, Frick M, Mair N,Dietl P: Threshold calcium levels for la-mellar body exocytosis in type IIpneumocytes. Am J Physiol1999;277:L893-L900.

100 Pian MS, Dobbs LG, Duzgunes N: Posi-tive correlation between cytosolic freecalcium and surfactant secretion in cul-tured rat alveolar type II cells. BiochimBiophys Acta 1988;960:43-53.

101 Chow RH, von Ruden L, Neher E: Delayin vesicle fusion revealed byelectrochemical monitoring of single se-cretory events in adrenal chromaffincells. Nature 1992;356:60-63.

102 Chow RH, Klingauf J, Neher E: Timecourse of Ca2+ concentration triggeringexocytosis in neuroendocrine cells. ProcNatl Acad Sci U S A 1994;91:12765-12769.

103 Chernomordik LV, Kozlov MM: Mem-brane hemifusion: crossing a chasm intwo leaps. Cell 2005;123:375-382.

104 Wong JL, Koppel DE, Cowan AE, WesselGM: Membrane hemifusion is a stableintermediate of exocytosis. Dev Cell2007;12:653-659.

105 Zampighi GA, Zampighi LM, Fain N,Lanzavecchia S, Simon SA, Wright EM:Conical electron tomography of a chemi-cal synapse: vesicles docked to the ac-tive zone are hemi-fused. Biophys J2006;91:2910-2918.

106 Miklavc P, Albrecht S, Wittekindt OH,Schullian P, Haller T, Dietl P: Existenceof exocytotic hemifusion intermediateswith a lifetime of up to seconds in typeII pneumocytes. Biochem J 2009;424:7-14.

107 Jahn R, Lang T, Sudhof TC: Membranefusion. Cell 2003;112:519-533.

108 Pian MS, Dobbs LG: Activation of G pro-teins may inhibit or stimulate surfactantsecretion in rat alveolar type II cells.Am J Physiol 1994;266:L375-L381.

109 Sano K, Voelker DR, Mason RJ: Effectof secretagogues on cytoplasmic freecalcium in alveolar type II epithelial cells.Am J Physiol 1987;253:C679-C686.

110 Griese M, Gobran LI, Rooney SA: Sig-nal-transduction mechanisms of ATP-stimulated phosphatidylcholine secretionin rat type II pneumocytes: interactionsbetween ATP and other surfactantsecretagogues. Biochim Biophys Acta1993;1167:85-93.

111 Geppert M, Goda Y, Hammer RE, Li C,Rosahl TW, Stevens CF, Sudhof TC:Synaptotagmin I: a major Ca2+ sensor fortransmitter release at a central synapse.Cell 1994;79:717-727.

112 Pang ZP, Melicoff E, Padgett D, Liu Y,Teich AF, Dickey BF, Lin W, Adachi R,Sudhof TC: Synaptotagmin-2 is essen-tial for survival and contributes to Ca2+

triggering of neurotransmitter release incentral and neuromuscular synapses. JNeurosci 2006;26:13493-13504.

113 Davis CW, Dickey BF: Regulated airwaygoblet cell mucin secretion. Annu RevPhysiol 2008;70:487-512.

114 Caohuy H, Srivastava M, Pollard HB:Membrane fusion protein synexin(annexin VII) as a Ca2+/GTP sensor inexocytotic secretion. Proc Natl Acad SciU S A 1996;93:10797-10802.

115 Sen N, Spitzer AR, Chander A: Calcium-dependence of synexin binding may de-termine aggregation and fusion of lamel-lar bodies. Biochem J 1997;322:103-109.

116 Liu L, Chander A: Stilbene disulfonic ac-ids inhibit synexin-mediated membraneaggregation and fusion. Biochim BiophysActa 1995;1254:274-282.

117 Chander A, Sen N, Spitzer AR: Synexinand GTP increase surfactant secretion inpermeabilized alveolar type II cells. AmJ Physiol Lung Cell Mol Physiol2001;280:L991-L998.

118 Chander A, Wu RD: In vitro fusion oflung lamellar bodies and plasma mem-brane is augmented by lung synexin.Biochim Biophys Acta 1991;1086:157-166.

119 Chander A, Sen N, Naidu DG, Spitzer AR:Calcium ionophore and phorbol esterincrease membrane binding of annexina7 in alveolar type II cells. Cell Calcium2003;33:11-17.

120 Mair N, Haller T, Dietl P: Exocytosis inalveolar type II cells revealed by cell ca-pacitance and fluorescence measure-ments. Am J Physiol 1999;276:L376-L382.

121 Muallem S, Kwiatkowska K, Xu X, YinHL: Actin filament disassembly is a suf-ficient final trigger for exocytosis innonexcitable cells. J Cell Biol1995;128:589-598.

122 Holt M, Riedel D, Stein A, Schuette C,Jahn R: Synaptic vesicles are constitu-tively active fusion machines that func-tion independently of Ca2+. Curr Biol2008;18:715-722.

123 Rose F, Kurth-Landwehr C, Sibelius U,Reuner KH, Aktories K, Seeger W,Grimminger F: Role of actindepolymerization in the surfactant se-cretory response of alveolar epithelialtype II cells. Am J Respir Crit Care Med1999;159:206-212.

124 Malacombe M, Bader MF, Gasman S:Exocytosis in neuroendocrine cells: newtasks for actin. Biochim Biophys Acta2006;1763:1175-1183.

125 Ehre C, Rossi AH, Abdullah LH, De PK,Hill S, Olsen JC, Davis CW: Barrier roleof actin filaments in regulated mucin se-cretion from airway goblet cells. Am JPhysiol Cell Physiol 2005;288:C46-C56.

126 Chernomordik L, Chanturiya A, Green J,Zimmerberg J: The hemifusion interme-diate and its conversion to complete fu-sion: regulation by membrane composi-tion. Biophys J 1995;69:922-929.

127 Zimmerberg J, Chernomordik LV: Neu-roscience. Synaptic membranes bend tothe will of a neurotoxin. Science2005;310:1626-1627.


12

128 Kooijman EE, Chupin V, de KB, BurgerKN: Modulation of membrane curvatureby phosphatidic acid andlysophosphatidic acid. Traffic2003;4:162-174.

129 Zeniou-Meyer M, Zabari N, Ashery U,Chasserot-Golaz S, Haeberle AM, DemaisV, Bailly Y, Gottfried I, Nakanishi H,Neiman AM, Du G, Frohman MA, BaderMF, Vitale N: Phospholipase D1 produc-tion of phosphatidic acid at the plasmamembrane promotes exocytosis of largedense-core granules at a late stage. J BiolChem 2007;282:21746-21757.

130 Williams MC: Conversion of lamellarbody membranes into tubular myelin inalveoli of fetal rat lungs. J Cell Biol1977;72:260-277.

131 Stahlman MT, Gray MP, Falconieri MW,Whitsett JA, Weaver TE: Lamellar bodyformation in normal and surfactant pro-tein B-deficient fetal mice. Lab Invest2000;80:395-403.

132 Haller T, Dietl P, Stockner H, Frick M,Mair N, Tinhofer I, Ritsch A, EnhorningG, Putz G: Tracing surfactant transfor-mation from cellular release to insertioninto an air-liquid interface. Am J PhysiolLung Cell Mol Physiol 2004;286:L1009-L1015.

133 Miklavc P, Wittekindt OH, Felder E,Dietl P: Ca2+-dependent actin coatingof lamellar bodies after exocytotic fu-sion: a prerequisite for content releaseor kiss-and-run. Ann N Y Acad Sci2009;1152:43-52.

134 Singer W, Frick M, Haller T, Bernet S,Ritsch-Marte M, Dietl P: Mechanicalforces impeding exocytotic surfactant re-lease revealed by optical tweezers.Biophys J 2003;84:1344-1351.

135 Tsilibary EC, Williams MC: Actin in pe-ripheral rat lung: S1 labeling and struc-tural changes induced by cytochalasin. JHistochem Cytochem 1983;31:1289-1297.

136 van Weeren L, de Graaff AM, JamiesonJD, Batenburg JJ, Valentijn JA: Rab3Dand actin reveal distinct lamellar bodysubpopulations in alveolar epithelial typeII cells. Am J Respir Cell Mol Biol2004;30:288-295.

137 Finkelstein A, Zimmerberg J, Cohen FS:Osmotic swelling of vesicles: its role inthe fusion of vesicles with planar phos-pholipid bilayer membranes and its pos-sible role in exocytosis. Annu Rev Physiol1986;48:163-174.

138 Breckenridge LJ, Almers W: Final stepsin exocytosis observed in a cell with gi-ant secretory granules. Proc Natl AcadSci U S A 1987;84:1945-1949.

139 Wright JR, Clements JA: Metabolism andturnover of lung surfactant. Am RevRespir Dis 1987;136:426-444.

140 Yu HY, Bement WM: Control of localactin assembly by membrane fusion-de-pendent compartment mixing. Nat CellBiol 2007;9:149-159.


review cellular physiology · 2009-11-27 · 2 provided by electron microscopy (em) studies [10]....

Documents