Regulation of surfactant secretion

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 Ca²⁺-regulated mechanism that likely results in the activation of Ca²⁺-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 and/or 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.

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 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 contribute 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 finally 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, Ikegami et al., 1994) and tissue culture (Osanai et al., 1998) experiments suggest that newly synthesized 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 β-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 (PKC) and ionophores (ionomycin and A23187) that promote Ca²⁺ influx into the cell (Chander and Fisher, 1990, Wright and Dobbs, 1991, Mason and Voelker, 1998, Rooney, 1998).