Calcium‐PS‐dependent protein kinase C and surfactant protein A in isolated fetal rabbit type II alveolar cells and surfactant‐related material

Abstract

Summary. The fetal lung secretes significant quantities of surfactant during late gestation to prepare for initiation of respiration at birth. However, the mechanism by which this occurs has not been determined. Since Ca 2+ -phosphatidylserine (PS)-dependent protein kinase C has been implicated in surfactant secretion in adult lung, the present study was done to determine whether this enzyme is also involved in the initiation of surfactant release from fetal type II cells. Type II cells isolated from gestational day-24 fetal rabbits were used. Cells were prelabelled with [32 P] and [3 H]choline and exposed to 4␤ phorbol ester (10 −5 M) for 2 h. Secretion product and subcellular fractions were isolated by removing the culture medium, mixing with homogenate from adult rabbit lung, and subfractionating by centrifugation on a sucrose gradient. Samples of secretion product were also prepared for electron microscopy. Ca 2+ -PS-dependent protein kinase C was also assayed in some samples, and an add-back technique was used to determine whether enzyme activity in the intracellularly stored surfactant fraction was due to contamination.The results showed that material released by fetal type II cells after exposure to phorbol ester coprecipitated with adult rabbit lung lamellar bodies and microsomes. Morphologically, a range of forms, including lamellar-body-like structures, was detected. The released material originated largely from the lamellar body compartment of the fetal type II cells and displayed immunoreactivity with antibody to surfactant protein A (SP-A) at 35 and 70 kDa apparent molecular mass. Assay of protein kinase C in fetal type II cells showed that exposure to conditioned medium, which induces differentiation, increased activity. Incubation with phorbol ester induced translocation of activity to the microsomal fraction. Add-back assays suggested that protein kinase C activation by treatment with phorbol ester induced translocation of enzyme activity to the lamellar body fraction; none was detected prior to treatment.These results support a role for Ca 2+ -PS-dependent protein kinase C in initiation of surfactant release by interaction with the developing lamellar body compartment in fetal type II cells.

Regulation of surfactant secretion in alveolar type II cells

“…PKC substrates in this pathway have not been identified. Phorbol ester-induced translocation of PKC to the LBs has been reported (125), suggesting that it might phosphorylate proteins located at or near LBs. One likely candidate may be annexin VII (synexin), which has been implicated in Ca 2ϩ -dependent LB fusion with the plasma membrane (see Ref.…”

Dopamine activates ERKs in alveolar epithelial cells via Ras-PKC-dependent and Grb2/Sos-independent mechanisms

“…PKC proteins control many ATII cell functions, including regulation of surfactant phospholipid secretion (29,48), modulation of arachidonic acid metabolism (43), or stimulation of ion transporters (61). Nevertheless, there are no studies linking PKC proteins to ERK activation in ATII cells, although a recent report has described the participation of PKC proteins in ERK activation in A549 cells (34).…”

Signal transduction events involved in TPA downregulation of SP-A gene expression

“…In rat type II cells, the phorbol ester TPA activates PKC-␣, -␤I, -␤II, -␦, and -and has been shown to target PKC-to a membrane fraction (14). In fetal rabbit type II cells, TPA causes the translocation of PKC activity to a lamellar body fraction (39). TPA stimulates PKC activity associated with a membrane fraction rather than the cytosolic fraction of lung epithelial cell homogenates (2).…”

“…Activation of PKC impairs the barrier function of epithelia and, in the lung, increases endothelial permeability and can lead to pulmonary edema and respiratory distress syndrome (RDS) (5,43). Several studies have documented the involvement of certain PKC isoforms, i.e., PKC-and Ca-/phosphatidylserine (PS)-dependent PKC, in surfactant secretion in primary cultures of alveolar type II cells (14,39).…”