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Review

Regulation of surfactant secretion

Seamus A. RooneyU
Di ision of Perinatal Medicine, Department of Pediatrics, Yale Uni ersity School of Medicine, P.O. Box 208064, New Ha en, CT 06520-8064, USA Received in revised form 15 November 2000; accepted 12 February 2001

Abstract Lung surfactant is synthesized in the alveolar type II cell. Its lipids and hydrophobic proteins SP-B and SP-C. are stored in lamellar bodies and secreted by regulated exocytosis. In contrast, the hydrophilic proteins SP-A and SP-D. appear to be secreted independently of lamellar bodies. Regulation of surfactant secretion is mediated by at least three distinct signaling mechanisms: activation of adenylate cyclase with formation of cAMP and activation of cAMP-dependent protein kinase; activation of protein kinase C; and a Ca2q-regulated mechanism that likely results in the activation of Ca2q-calmodulin-dependent protein kinase. These signaling mechanisms are activated by a variety of agonists, some of which may have a physiological role. ATP is one such agent and it activates all three signaling mechanisms. There is increasing information on the identity of several of the signaling proteins involved in surfactant secretion although others remain to be established. In particular the identity of the phospholipase C, protein kinase C and phospholipase D isomers expressed in the type II cell andror involved in surfactant secretion has been established. Distal steps in the secretory pathway beyond protein kinase activation as well as the physiological regulation of surfactant secretion, are major issues that need to be addressed. 2001 Elsevier Science Inc. All rights reserved.
Keywords: Lung surfactant; Secretion; Signal transduction; Type II pneumocyte; Protein kinase; Phospholipase D; Phospholipase C- 3; P2Y2 receptor

1. Introduction Lung surfactant consists largely of lipids with phosphatidylcholine PC. being by far the most abundant component van Golde et al., 1988; Rooney et al., 1994.. More than half of the PC in surfactant is disaturated Rooney, 1992. and it is

Corresponding author. Tel.: q1-203-785-4650; fax: q1203-785-7194. E-mail address: seamus.rooney@yale.edu S.A. Rooney..

disaturated PC that is critical for the biophysical role of surfactant Keough, 1998.. Surfactant also contains four unique proteins: surfactant protein A SP-A., SP-B, SP-C and SP-D Kuroki and Voelker, 1994; Johansson and Curstedt, 1997.. The type II pneumocyte is the cellular source of surfactant within the lung van Golde et al., 1988; Wright and Dobbs, 1991; Rooney et al., 1994.. The type II cell can synthesize all of the lipid and protein components of surfactant. SP-A, SP-B and SP-D are also synthesized in airway epithelial cells but the extent to which those cells con-

1095-6433r01r$ - see front matter 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 0 1 . 0 0 3 2 0 - 8

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tribute to surfactant production is not clear Johansson et al., 1994.. Surfactant secretion has been measured in several systems ranging from intact animals in vivo to isolated type II cells in culture Chander and Fisher, 1990; Wright and Dobbs, 1991; Mason and Voelker, 1998; Rooney, 1998.. Isolated type II cells have been the model of choice for most studies on the regulation of surfactant secretion. Studies on surfactant secretion have focused primarily on its lipid components, particularly PC or disaturated PC. Surfactant phospholipids are synthesized in the endoplasmic reticulum and stored in lamellar inclusion bodies, the secretory organelle characteristic of the type II cell, and nally secreted into the alveolar lumen by the process of regulated exocytosis Mason and Voelker, 1998; Rooney, 1998; Rooney et al., 1994.. Lamellar bodies in the process of exocytosis have been captured by electron microscopy Fig. 1.. The phospholipid composition of isolated lamellar bodies is virtually identical to that of surfactant Rooney, 1992. and it is clear that surfactant phospholipids are secreted together with lamellar bodies Chander and Fisher, 1990; Wright and Dobbs, 1991; Mason and Voelker, 1998; Rooney, 1998.. It is possible that there is also some constitutive secretion of surfactant lipids; the basal secretion of phospholipids that is observed in isolated type II cells cultured without secretagogues may well be a constitutive process. Secretion of surfactant proteins has been less extensively investigated. Lamellar bodies are enriched in SP-B and SP-C Oosterlaken-Dijksterhuis et al., 1991. and it is likely that these hydrophobic proteins are secreted together with the phospholipids Rooney, 1998.. Indeed in recent preliminary experiments, we have found that secretion of SP-B and SP-C is stimulated by the surfactant phospholipid secretagogue 12-O-tetradecanoylphorbol-13-acetate TPA. in primary cultures of adult rat type II cells. Secretion of SP-A appears to be largely constitutive and not regulated Mason and Voelker, 1998; Rooney, 1998.. SP-A secretion in isolated type II cells is not generally stimulated by agonists that stimulate PC secretion Froh et al., 1993; Rooney et al., 1993; Xu et al., 1998., although in two studies it was stimulated by TPA Dobbs et al., 1982; Xu et al., 1998.. Furthermore, both in vivo Ikegami et al., 1992, 1994. and tissue culture Osanai et al., 1998. experiments suggest that newly synthesized

Fig. 1. A lamellar body in the process of exocytosis from a type II cell. Reproduced with permission from Rooney et al. 1994..

SP-A is secreted independently of lamellar bodies. Lamellar bodies are devoid of SP-D Crouch et al., 1991; Voorhout et al., 1992. so that protein must also be secreted independently of the lipids. Indeed secretion of SP-D was not stimulated by TPA or terbutaline in cultured type II cells Xu et al., 1998.. In summary, secretion of surfactant phospholipids and SP-B and SP-C occurs largely by regulated exocytosis of lamellar bodies whereas secretion of SP-A and SP-D occurs by a different mechanism and may be mainly constitutive. A variety of physiological and pharmacological agents stimulate surfactant PC secretion in isolated type II cells and there are also agents that inhibit it Chander and Fisher, 1990; Wright and Dobbs, 1991; Mason and Voelker, 1998; Rooney, 1998.. Surfactant secretagogues include those that activate cell surface receptors as well as those that penetrate the cell and activate downstream signaling steps Fig. 2.. Well established PC secretagogues in cultured type II cells include -adrenergic and adenosine A 2B receptor agonists, P2Y2 receptor agonists ATP, UTP., forskolin and cholera toxin that directly or indirectly activate adenylate cyclase AC., agents such as TPA and cell-permeable diacylglycerols DAGs. that directly activate protein kinase C

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Fig. 2. Schematic representation of signal transduction mechanisms mediating surfactant phospholipid secretion in type II cells. See text for details. Adapted from Rooney et al. 1994.. Abbreviations: AC, adenylate cyclase; CaCM PK, calciumrcalmodulin-dependent protein kinase; DAG, diacylglycerol; IP3 , inositol trisphosphate; PA, phosphatidic acid; PKA, protein kinase A cAMP-dependent protein kinase.; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; TPA, 12-O-tetradecanoylphorbol-13-acetate.

PKC. and ionophores ionomycin and A23187. that promote Ca2q inux into the cell Chander and Fisher, 1990; Wright and Dobbs, 1991; Mason and Voelker, 1998; Rooney, 1998..

2. Signaling mechanisms of surfactant secretion A working model of the signal transduction mechanisms that mediate surfactant secretion is shown in Fig. 2. The signaling mechanisms consist of three distinct pathways although there is overlap and interactions among them Griese et al., 1993; Rooney, 1998.. The rst mechanism involves the activation of AC, generation of cyclic AMP cAMP. and subsequent activation of cAMP-dependent protein kinase protein kinase A, PKA.. This pathway is activated by -adrenergic and adenosine A 2B receptors that are coupled to AC via the heterotrimeric GTP-binding protein G-protein. Gs . It is also activated by ATP acting at an unidentied AC-coupled receptor that is distinct from the adenosine A 2B receptor Gobran and Rooney, 1997; Rooney, 1998.. The AC pathway is also activated by secretagogues that bypass the receptors: cholera toxin,

which permanently activates Gs , and forskolin, which directly activates AC. The second mechanism involves direct or indirect activation of PKC. TPA and cell permeable DAGs directly activate PKC. ATP and UTP bind to P2Y2 receptors that are coupled to phospholipase C PLC.- 3 via Gq . Activation of PLC- 3 results in the hydrolysis of phosphatidylinositol bisphosphate and the formation of DAG and inositol trisphosphate IP3 .. DAG activates PKC which in turn activates phospholipase D PLD.. PLD hydrolysis of PC leads to the formation of choline not shown in Fig. 2. and phosphatidic acid PA.. Phosphatidate phosphatase not shown. converts PA to DAG, which then further activates PKC. It is also possible that PA itself has a signaling function Hodgkin et al., 1998; Wakelam, 1998.. In some systems the adenosine A 2B receptor is also coupled to PLC via Gq Nyce, 1999., but there is no evidence that adenosine agonists activate this pathway in type II cells Griese et al., 1991.. The third mechanism involves elevation of intracellular Ca2q levels Chander and Fisher, 1990; Wright and Dobbs, 1991; Mason and Voelker, 1998; Rooney, 1998.. This can be accomplished by an increase in IP3 in response to PLC- 3 activa-

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tion; IP3 promotes mobilization of Ca2q from intracellular stores. It can also be accomplished by ionophores that promote Ca2q inux into the cell from the medium. Ca2q activates a Ca2qcalmodulin-dependent protein kinase CaCM-PK. and may also act synergistically with DAG to activate some PKC isoforms. Protein phosphorylation by PKA, PKC or CaCM PK ultimately leads to surfactant secretion.

3. Signaling proteins It is evident that there are several signaling proteins involved in the surfactant secretion signaling pathways. Many subtypes andror isomers of the receptors and other signaling proteins listed in Fig. 2 are known to exist. There is increasing information on the identity of the specic proteins involved in surfactant secretion. There is evidence that it is the 2 , A 2B and P2Y2 subtypes of the -adrenergic, adenosine and P2 receptors, respectively, that regulate surfactant secretion in type II cells. Recent studies have identied the isoforms of PLC, PKC and PLD that exist in the type II cell and that are likely to mediate agonist-stimulated surfactant secretion. However, the isoformsrsubtypes of AC, PKA and CaCMPK involved in surfactant secretion remain to be established. Of the three -receptors, 3 is not expressed in the lung Barnes, 1995., but the 1 and 2 genes are both expressed in the type II cell Isohama et al., 1995; Gobran et al., 1998.. However, pharmacological data suggest that surfactant secretion is regulated by the 2-receptor Fabisiak et al., 1987; Ewing et al., 1992.. There are four subtypes of adenosine receptors A 1 , A 2A , A 2B and A 3 Ralevic and Burnstock, 1998. and all four are expressed in the type II cell Gobran et al., 1998.. However, agonist and antagonist potency data and the fact that adenosine agonists that stimulate surfactant secretion also enhance cAMP formation, show that it is the adenosine A 2B receptor that regulates surfactant secretion Rooney, 1998.. P2 receptors are divided into two major groups: P2X receptors are ion channels; whereas P2Y are metabotropic receptors coupled to Gproteins Ralevic and Burnstock, 1998.. Presently, seven P2X and ve P2Y mammalian subtypes have been cloned Ralevic and Burnstock, 1998..

There is good evidence that the P2 purinoceptor responsible for stimulation of surfactant secretion is P2Y2 Rooney, 1998.. The P2Y2 receptor was termed P2u in an older nomenclature system Fredholm et al., 1994; Ralevic and Burnstock, 1998.. ATP and UTP are equally potent at P2Y2 receptors Ralevic and Burnstock, 1998. and both nucleotides stimulate surfactant secretion in type II cells with similar EC 50 values Gobran et al., 1994.. Additional agonist potency order data are also consistent with the P2Y2 receptor being involved in surfactant secretion. Griese et al., 1991; Gobran et al., 1994; Gilllan and Rooney, 1988.. The P2Y2 receptor is expressed in the type II cell Gobran et al., 1994; Rice et al., 1995; Gobran et al., 1998.. Indeed the rat P2Y2 receptor was rst cloned using a type II cell cDNA library Rice et al., 1995.. Ten mammalian PLC isoforms have been identied and based on amino acid sequence they are classied as , and types Rhee and Bae, .. PLC- , of which there are four isoforms, 1997 1, 2, 3 and 4, is the only one activated by G-protein coupled receptors Exton, 1994; Rhee and Bae, 1997. and, is therefore, the type most likely involved in surfactant secretion. We have found that PLC- 3 is the only PLC- isomer present in the type II cell Gobran et al., 1998. and must, therefore, be the one involved in regulation of surfactant secretion. Eleven PKC isoforms are currently known to exist Nishizuka, 1995; Rasmussen et al., 1995.. They include the Ca2q-dependent conventional , I, II and isoforms as well as the novel , , and isoforms and the atypical , PKD in the mouse. and isoforms none of which require Ca2q for activation Rasmussen et al., 1995.. The , I, II, , , , and isoforms have been identied in the type II cell although PKC- is barely detectable Linke et al., 1997; Gobran et al., 1998.. We have established that the P2Y2 agonists ATP and UTP as well as TPA and DAG activate PKC- and that TPA and DAG also activate PKCs , I, II, and Gobran and Rooney, 1999.. TPA was also reported to activate PKCs , , and in another type II cell study in which PKC- was not examined and PKCs I and II were not distinguished Linke et al., 1997.. PKCwas not activated by any of the surfactant secretagogues examined in either study Linke et al., 1997; Gobran and Rooney, 1999..

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Fig. 3. RT PCR analysis of PLD mRNAs in adult rat lung tissue and in type II cells from adult and 1-day old newborn rats. RT PCR was carried out on total RNA as described previously Gobran et al., 1998., the products subjected to electrophoresis on agarose gels and visualized by staining with ethidium bromide. Forward and reverse primer sequences were 5 -AACAAGGTGTGCGGATTTTC-3 and 5 TGAAGTTGGCGATAGAGGCT-3 for PLD1 and 5 -TGGCTACAGTCAGCCCTTCT-3 and 5 -AGGGTTCTGTGGCATAGTGG-3 for PLD2. The PLD1 and PLD2 product sizes were 472 and 414 bp, respectively. Lanes 1 and 2, adult lung tissue; lanes 3 and 4, adult type II cells; lanes 5 and 6, newborn type II cells.

and RII . and three of the catalytic C , C and C . subunit Rubin, 1994.. There is no information on which PKA isoforms. exist in the type II cell. There are at least three CaCM PKs: CaMKI, CaMK-II and CaMK-IV as well as myosin light chain kinase Hanks and Hunter, 1995; Means, 2000.. CaMK-II has 4 isomers , , and Hanks and Hunter, 1995.. There is currently no information on the identity of the CaCM PKs in the type II cell or those that regulate surfactant secretion. However, KN-62, supposedly a specic inhibitor of CaMK-II Liu, 1998., completely abolished the stimulatory effect of A23187 and decreased that of ATP on PC secretion in type II cells Liu, 1998..

There are two mammalian PLDs, PLD1 and PLD2 Waite, 1999.. Based on the DNA sequences of PLD1 Park et al., 1997. and PLD2 Kodaki and Yamashita, 1997., we recently measured expression of both genes in the lung by RT-PCR. As shown in Fig. 3, both PLD1 and PLD2 are expressed in type II cells and levels of expression are the same in lung tissue and type II cells. Two splice variants of rat Katayama et al., 1998. and human Hammond et al., 1997. PLD1 have been cloned; PLD1a and PLD1b are identical except for a 38 amino acid deletion in PLD1b Exton, 1998.. We used primers designed to amplify both PLD1a and PLD1b so we do not know whether one or both variants are expressed in the type II cell. In keeping with the reportedly low levels of PLD in other systems, we were unable to detect either PLD1 or PLD2 using a commercial rabbit polyclonal antibody. These data show that both PLDs are expressed in the type II cell. However, the PLD isomers. involved in surfactant secretion remain to be established. There are nine mammalian AC isoforms and most are expressed in the lung Sunahara et al., 1996.. AC-II and AC-IV are expressed in the type II cell Pian and Dobbs, 1995. whereas AC-I is not expressed in either the lung or type II cell Pian and Dobbs, 1995; Sunahara et al., 1996.. It is not known if other ACs exist in the type II cell. The AC isoforms. involved in surfactant secretion have not been identied. PKA consists of regulatory and catalytic subunits and there are four isoforms of the regulatory RI , RI , RII

4. Other signaling mechanisms In addition to those discussed, other signaling mechanisms may also be involved in surfactant secretion. For instance, there are data suggesting involvement of phospholipase A 2 as well as a PC-specic PLC in ATP and TPA mediated secretion Rooney, 1998.. High- and low-density serum lipoproteins were reported to stimulate PC secretion by activation of receptors coupled via Gi to the PKC signaling mechanism VoynoYasenetskaya et al., 1993; Pian and Dobbs, 1997.. Mastoparan, an agent that activates several signaling mechanisms, was reported to be a powerful surfactant secretagogue and there were indications that its effect is also mediated in part by Gi Joyce-Brady et al., 1991.. In addition to those discussed here, a number of other surfactant secretagogues have been reported Rooney, 1998.. However, some of those ndings were not conrmed. The bisindolyl maleimide Ro-318220 antagonizes the effects of different surfactant secretagogues in type II cells Rooney and Gobran, 1997.. Because its IC 50 values are similar for the different secretagogues it is likely that the effect of Ro-318220 is due to inhibition of a common signal transduction step Rooney and Gobran, 1997.. Although Ro-318220 is known to inhibit PKC Davis et al., 1989; Way et al., 2000., it has also been reported to inhibit steps in the mitogen activated protein MAP. kinase signaling pathway Beltman et al., 1996; Alessi, 1997.. There is

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evidence that the MAP-kinase pathway is activated by P2Y2 agonists in a number of different cell types Huwiler and Pfeilschifter, 1994; Graham et al., 1996; Boarder and Hourani, 1998; Soltoff et al., 1998; Gao et al., 1999. and it can also be activated by ionophores, TPA and other receptors coupled to G-proteins Seger and Krebs, 1995., including the adenosine A 2B receptor Gao et al., 1999.. We, therefore, considered the possibility that the MAP kinase signaling cascade is distal to protein kinase activation in all three signaling mechanisms regulating surfactant secretion Fig. 2. and that the effect of Ro-318220 is due to inhibition of the MAP kinase system. We examined the effect of MAP kinase inhibitors on surfactant secretion in response to terbutaline, the adenosine receptor agonist 5 -N-ethylcarboxyamidoadenosine NECA., ATP, TPA and ionomycin. We used three inhibitors, PD-98059, SB-203580 and SB-202190, that are known to inhibit steps in the MAP kinase signaling cascade Cohen, 1997.. We used 10y5 M NECA, 10y5 M ATP, 10y7 M TPA, 3.5= 10y5 M terbutaline and 5 = 10y8 M ionomycin and preincubated the cells with the inhibitors 10y5 M. for 30 min before addition of the agonists. None of the inhibitors, however, decreased the stimulatory effects of the surfactant secretagogues examined. The inhibitors on their own, had no effect on PC secretion. These data, therefore, do not support a role for the MAP kinase signaling cascade in surfactant secretion. Edwards et al. 1998. came to the same conclusion. They found that although ATP and TPA activated the MAP kinase cascade, MAP kinase inhibitors did not diminish the effects of those agonists on surfactant secretion Edwards et al., 1998.. Involvement of the MAP kinase cascade in SPA inhibition of surfactant secretion was also discounted Mason and Voelker, 1998.. Tyrosine phosphorylation is often upstream of the MAP kinase cascade and there is evidence that tyrosine kinases are activated by P2Y receptors Boarder and Hourani, 1998.. Tyrosine phosphorylation is another potential step that might be inhibited by Ro-318220. Therefore, we examined the effects of tyrosine kinase inhibitors on surfactant secretion in response to terbutaline, NECA, ATP, TPA and ionomycin. The experimental design was as described above and we used the broad-based tyrosine kinase inhibitors tyrphostins A23 and A25, and genistein 10y5 M..

None of the inhibitors decreased the effects of any of the surfactant secretagogues examined. These data, therefore, do not support a role for tyrosine phosphorylation in surfactant secretion.

5. Distal steps in surfactant secretion Distal steps in the surfactant secretory pathway have received little attention. As shown in Fig. 2, all of the signal transduction pathways are believed to result in phosphorylation of proteins following activation of specic protein kinases. There is virtually no information on the identity of the proteins phosphorylated in response to surfactant secretagogues although TPA Warburton et al., 1991. and terbutaline Zimmerman et al., 1996. were reported to promote phosphorylation of a number of proteins in type II cells. Relatively little is known of the steps beyond protein phosphorylation that ultimately result in exocytosis of lamellar bodies Rooney, 1998.; there is essentially no information on how the SNARE paradigm of vesicle lamellar body. docking and membrane fusion Bajjalieh and Scheller, 1995; Thiel, 1995; Pelham, 1999. applies to surfactant secretion in type II cells Rooney, 1998..

6. Physiological regulation of surfactant secretion Little is known about physiological regulation of surfactant secretion. -Agonists Oyarzun and Clements, 1978; Abdellatif and Hollingsworth, 1980., cholinergic agonists Oyarzun and Clements, 1977; Abdellatif and Hollingsworth, 1980; Rooney and Gobran, 1988. and adenosine Ekelund et al., 1985. stimulate surfactant secretion in vivo. However, the mere fact that an agent has an effect in vivo does not establish that it has a physiological role. Blockade of a physiological response, on the other hand, can yield information on the pharmacological mechanism involved. Two physiological factors are known to stimulate surfactant secretion in vivo: ventilation, which stimulates secretion in both adults Oyarzun and Clements, 1977, 1978. and newborns Lawson et al., 1979; Rooney and Gobran, 1988., and labor, which stimulates secretion in newborns Rooney et al., 1977.. At least part of the effect of ventilation may be due to a direct mechanical effect on the type II cell as stretch increases surfactant

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secretion in cells cultured on a stretchable membrane Wirtz and Dobbs, 1990.. There is evidence that the AC signaling mechanism is involved in the physiological regulation of surfactant secretion. A physiological role for receptor activation is suggested by the fact that the stimulatory effects of ventilation Oyarzun and Clements, 1978. and labor Marino and Rooney, 1981. are antagonized by -antagonists. Inhibitors of prostaglandin synthesis also antagonize the stimulatory effects of labor Marino and Rooney, 1981. and ventilation Oyarzun and Clements, 1978; Rooney and Gobran, 1988. suggesting a physiological role for prostaglandins. Prostaglandins Anderson et al., 1978. and prostacyclin Rose et al., 1999. have been reported to stimulate surfactant secretion in type II cells and the effect of prostacyclin was accompanied by an increase in cAMP Rose et al., 1999. suggesting that the effect is mediated by the AC pathway. A physiological role for the adenosine A 2B or ACcoupled ATP receptor is suggested by the fact that the ventilation induced increase in surfactant secretion in newborn rabbits is attenuated by 8-phenyltheophylline Rooney and Gobran, 1988.. Finally, atropine blocks the effect of ventilation in vivo Oyarzun and Clements, 1977; Lawson et al., 1979; Rooney and Gobran, 1988. suggesting cholinergic regulation. However, there is substantial evidence that cholinergic regulation of surfactant secretion is mediated by activation of -receptors on the type II cell in response to catecholamines released from the adrenal medulla Rooney, 1998.. Whether the PKC and CaCM PK pathways have a physiological role is less clear. Apart from ATP, UTP and possibly lipoproteins Pian and Dobbs, 1997., the only well established surfactant secretagogues that activate the PKC signaling pathway are non-physiological agonists such as TPA and cell permeable DAGs while those that activate the CaCM PK pathway are the equally unphysiological ionophores ionomycin and A23187. It is highly likely that some physiological agonist activates those signaling mechanisms and it is tempting to speculate that ATP or UTP may be such an agonist. Currently, there is no information on whether the P2Y2 receptor on the type II cell has a physiological role in the regulation of surfactant secretion, but there is good evidence that the P2Y2 receptor has a physiological role in many other systems Ralevic and Burnstock,

1998.. Based on tissue culture data, ATP levels in bronchoalveolar lavage uid Rice et al., 1989. are sufcient to stimulate surfactant secretion. The stretch induced increase in surfactant secretion in type II cells is accompanied by an increase in cellular Ca2q levels Wirtz and Dobbs, 1990. and a mechanical stimulus also promotes ATP release Grygorczyk and Hanrahan, 1997. and IP3 formation Felix et al., 1996. in airway epithelial cells and UTP release in astrocytes Lazarowski et al., 1997.. In the latter system, the released UTP activated P2Y receptors on the same cells Lazarowski et al., 1997.. Those data raise the possibility that the stimulatory effect of stretch, and hence ventilation, on surfactant secretion is mediated by the type II cell P2Y2 receptor and, therefore, by the PKC andror CaCM PK signaling pathways. Secretion is clearly an indispensable step in the overall homeostasis of surfactant Wright and Dobbs, 1991; Rooney et al., 1994.. Failure of secretion would likely lead to a drastic deciency in surfactant. Because severe surfactant deciency is probably incompatible with life, it is likely that there is sufcient redundancy so that a fatal defect in surfactant secretion does not occur. This may account for the multiple signaling mechanisms that are involved in the regulation of surfactant secretion.

Acknowledgements Work in the authors laboratory was supported by grants HL-31175 and HL-43320 from the National Institutes of Health. Previously unpublished data were generated in collaboration with Laurice I. Gobran and Zhi-xin Xu. References
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