Tetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential

doi:10.1016/0005-2736(87)90200-8

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 902, Issue 3, 3 September 1987, Pages 317-326

https://doi.org/10.1016/0005-2736(87)90200-8 Get rights and content

Abstract

Alveolar type II cells were isolated from adult rat lungs after tissue dissociation with elastase. The effect of known secretagogues on transmembrane potential was examined in freshly isolated cells (day 0 cells) and in cells after one day of primary culture (day 1 cells). Freshly isolated type II cells were incubated with 3,3′-dipentyloxacarbocyanine (di-O-C₅(3)) or 3,3′-dipropylthiadicarbocyanine (di-S-C₃(5)), dyes whose intracellular fluorescence intensity is a direct function of the cellular transmembrane potential. Fluorescence was continuously recorded by fluorescence spectrophotometry. Type II cells rapidly incorporated the dyes, and the addition of gramicidin (1 μg/ml) depolarized the cells as indicated by a change in fluorescence. Neither 12-O-tetradecanoylphorbol 13-acetate (TPA) nor terbutaline plus 3-isobutyl-1-methylxanthine (IBMX), which stimulate surfactant secretion from isolated alveolar type II cells, changed the transmembrane potential. The lipophilic cation triphenylmethylphosphonium (TPMP⁺) was used to quantitate the transmembrane potential of type II cells cultured for one day. Addition of TPA or terbutaline plus IBMX induced surfactant secretion but did not alter the transmembrane potential. To study further the relationship of secretion to the transmembrane potential, secretion was also determined in the presence of high extracellular potassium which depolarizes the cells and in the presence of choline in place of sodium. High potassium enhanced the basal secretion of phosphatidylcholine from 1.8% to 3.4% (P < 0.01, n = 7). Substitution of sodium chloride by choline chloride had no effect on basal secretion but enhanced TPA-induced secretion (P < 0.01). We conclude that high extracellular potassium induces membrane depolarization and stimulates surfactant secretion, but TPA or terbutaline plus IBMX stimulates secretion without detectable membrane depolarization and stimulation of secretion by TPA does not require extracellular sodium.

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Surfactant secretion is a critical regulated process in the metabolism of pulmonary surfactant. Presumably, because this process is vital to the survival of the organism, there are several independent pathways for stimulating secretion which work through different cell surface receptors and signaling mechanisms. In addition, there is apparent homeostatic regulation in that two components of surfactant, namely SP-A and dipalmitoylphosphatidylcholine, can inhibit secretion. Although secretion of surfactant has been studied for over two decades, there remains some important issues to be resolved. In vivo secretion can be stimulated by hyperventilation or even a single large breath. However, we do not know the biochemical mechanism for this physiologically important form of stimulation. In vitro, we know many of the proximal events in signaling, but we do not know how the lamellar bodies move within a cell or the docking mechanism at the plasma membrane. Many investigators have demonstrated that SP-A will inhibit secretion in vitro, but the mechanism is not known. Finally, there is a route of secretion of SP-A independent of lamellar bodies, but we do not know details of this pathway nor its regulation.
Ca<sup>+2</sup>-phosphatidylserine-dependent protein kinase C activfty in fetal, neonatal and adult rabbit lung and isolated lamellar bodies
1995, Life Sciences
Evidence indicates that Ca⁺²-phosphatidylserine-dependent protein kinase C (PKC) is involved in regulation of surfactant secretion. This study was done to examine PKC activity in lung as surfactant synthesis and secretion is initiated, and at birth and to compare these enzyme levels with those in the adult lung. NZW rabbits were used. Fetal and adult lungs were fractionated into subcellular compartments including a lamellar body fraction, which represents intracellular surfactant. The time course for microsomal enzyme activity was compared between 24th gestational day and adult rabbit lung. The reactivity appeared similar in both fractions. PKC specific activity displayed a prominent peak between the 27th and 30th gestational days in total homogenate and lamellar bodies. Specific activity was also high in nuclear, mitochondrial and microsomal fractions the day prior to birth. Adult levels were similar or higher. Total PKC activity was high during late gestation but declined sharply the day prior to birth. A marked increase was present on the first postnatal day. In contrast lamellar bodies displayed a peak in activity between the 27th and 30th gestational days followed by a decline to adult levels. Delipidation of lamellar body fraction indicated that the high enzyme activity in this fraction on the 27th gestational day was not artifactual. The changes observed in PKC in fetal, neonatal and adult lung indicate this enzyme activity changes in lung during the period of onset of surfactant synthesis and secretion during late gestation and may be associated with lamellar bodies, in 27th gestational day fetal lung.
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Undifferentiated type II alveolar cells were isolated from the fetal rabbit lung on the 24th gestational day, grown in vitro for 2–3 days, and used to test the hypothesis that activation of protein kinase C by 4β-phorbol ester (TPA) or the diacylglycerol analogue, sn-1-oleoyl-2-acetylglycerol (OAG), stimulates disaturated phosphatidylcholine (DSPC) synthesis and secretion. To measure secretion, cells were prelabelled with [³H]choline in serum-free medium or medium with 10% carbon-stripped fetal bovine serum for 24 h. The radiolabel was removed and TPA (10⁻⁶–10⁻⁹ M) or OAG (125, 250 or 500 μM) was incubated with the cells for 2 h. The medium was removed and filtered. Fresh medium with the same compound was added for an additional 16 h. To measure synthesis, cells were incubated with [³H]choline and concurrently TPA or OAG was added. Cells were removed at 2 or 18 h. After 2 h at concentrations of 10⁻⁸ M, TPA augmented the release of ³H-labelled DSPC, the major component of the surfactant, by cells incubated in serum-free medium. In the presence of carbon-stripped fetal bovine serum, TPA (10⁻⁷ and 10⁻⁶ M) induced release of DSPC. The incorporation of [³H]choline into intracellular DSPC was increased after 2 or 18 h in fetal alveolar cells exposed to TPA at 10⁻⁹ M or higher. OAG also significantly stimulated the release of labelled DSPC after 2 h at all concentrations tested. In contrast, OAG-exposed cells displayed a reduction of [³H]choline incorporation into cellular DSPC. Characterization of radioactive material released by prelabelled fetal type II cells showed that phorbol ester stimulation increased the release of material which co-migrated with adult rabbit lung lamellar bodies on a sucrose gradient. Electrophoretic examination of [γ-³²P]ATP phosphorylation sites in fetal type II cells showed that TPA and OAG induced an increase in phosphorylation of a group of proteins with apparent molecular masses of 45, 50 and 55 kDa. Addition of phosphatidylserine to the incubations produced substantial increases in the phosphorylation of these proteins, particularly in the presence of TPA. Fetal type II cells also displayed a phosphorylation product with an apparent molecular mass of 97 kDa. This protein as well as two high-molecular-mass products appeared to be particular to cells incubated with TPA plus phosphatidylserine and may in part account for the different action of TPA compared to OAG with regard to synthesis and secretion of DSPC by the fetal type II cells.
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^∗: Present address: Department of Pediatrics, Kobe University School of Medicine, Kobe, 650, Japan.

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Regular paperTetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential

Abstract

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Am. Rev. Respir. Dis.

J. Clin. Invest.

Lab. Invest.

Regular paper
Tetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential