Surfactant secretagogue activation of protein kinase C isoforms in cultured rat type II cells

1999 ◽  
Vol 277 (2) ◽  
pp. L251-L256 ◽  
Author(s):  
Laurice I. Gobran ◽  
Seamus A. Rooney
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Lung Surfactant ◽  
Type Ii Cells ◽  
Type Ii ◽  
Adult Rats ◽  
Activation Patterns ◽  
Physiological Regulation ◽  
Kinase C ◽  
Pkc Activation

Several lung surfactant secretagogues are known to activate protein kinase C (PKC) in type II cells. Such agents include 12- O-tetradecanoylphorbol 13-acetate (TPA) and cell-permeable diacylglycerols that directly activate PKC. Other agents include ATP and UTP, which act at P2Y2 receptors coupled to phosphoinositide-specific phospholipase C, activation of which leads to formation of diacylglycerols and consequent activation of PKC. Activation of PKC is associated with redistribution of enzyme from a cytosolic to a membrane fraction of the cell. We examined the PKC isomers that are translocated by ATP, UTP, TPA, and dioctanoylglycerol in cultured type II cells isolated from adult rats. PKC isoforms were identified by Western blotting using isoform-specific antibodies. Treatment of type II cells with ATP, UTP, TPA, and dioctanoylglycerol resulted in a significant redistribution of PKC-μ from cytosol to membrane. TPA and dioctanoylglycerol also activated PKC-α, -βI, -βII, -δ, and -η, but those isoforms were not activated by ATP or UTP. The effects of TPA and dioctanoylglycerol on PKC-μ were more pronounced than those of the P2Y2agonists, and the effect of TPA was also more rapid than that of ATP. The data show that direct activators and agents that generate endogenous diacylglycerols have different PKC activation patterns. Because it is activated by different types of secretagogues, PKC-μ may have an important role in the physiological regulation of surfactant secretion.

Stimulation of lung surfactant secretion by endothelin-1 from rat alveolar type II cells

1994 ◽  
Vol 266 (3) ◽  
pp. L255-L262 ◽  
Author(s):  
N. Sen ◽  
M. M. Grunstein ◽  
A. Chander
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Lung Surfactant ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Kinase C ◽  
Surfactant Secretion

Endothelin-1 (ET-1), a potent vasoactive peptide released by endothelial cells, alters cytosolic Ca2+ and phosphoinositide metabolism in cells of myogenic and nonmyogenic origin. We evaluated the effect of ET-1 on surfactant phosphatidylcholine (PC) secretion from alveolar type II cells labeled with [methyl-3H]choline. ET-1 stimulated the secretion of PC in a time- and dose-dependent manner. Binding of 125I-labeled ET-1 to type II cell membranes was saturable at approximately 1 nM and suggested the presence of a single type of receptor. The secretagogue effect of ET-1 was independently inhibited by nifedipine and nitrendipine, both L-type calcium channel blockers, and removal of extracellular calcium. ET-1 also increased cellular diacylglycerol content by approximately 50% within 30 s, which could not be attenuated by pretreatment with nifedipine, suggesting an early activation of protein kinase C that was independent of Ca2+ influx. Further, ET-1-stimulated PC secretion was also blocked by inhibitors of protein kinase C. Collectively, these results indicate that the binding of ET-1 to type II cells is coupled to the activation of protein kinase C, which increases calcium influx through L-type calcium channels, and results in increased secretion of lung surfactant. Since ET-1 is released from pulmonary micro- and macrovasculature endothelial cells in close proximity to type II cells, our findings support the novel concept that endothelial cells interact with alveolar type II cells in a paracrine fashion to regulate surfactant secretion.

Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells

1991 ◽  
Vol 261 (4) ◽  
pp. L105-L109
Author(s):  
T. A. Voyno-Yasenetskaya ◽  
L. G. Dobbs ◽  
M. C. Williams
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Pertussis Toxin ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Calmodulin Antagonist ◽  
Pi Turnover ◽  
Kinase C ◽  
Surfactant Secretion

Several different classes of agonists are known to stimulate exocytosis in type II cells. These agonists cause increases in second messengers, such as adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+, and/or stimulate protein kinase C. We studied generation of cAMP and phosphoinositide (PI) turnover in monolayer cultures of type II cells and measured [Ca2+]i in single cultured cells. ATP [10-4 M], which stimulates secretion of phosphatidylcholine (PC) and increases cellular cAMP, also stimulated PI turnover and increased [Ca2+]i. 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulates PC secretion and activates protein kinase C, did not increase [Ca2+]i. Pretreatment of type II cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the PC secretion induced by ATP and TPA and blocked the increase in PI turnover caused by ATP. ATP-dependent surfactant secretion and stimulation of PI turnover could also be inhibited by pretreatment of the cells with pertussis toxin. We used the fluorescent probe indo-1 to measure [Ca2+]i in single cultured type II cells. ATP produced rapid transient increases in [Ca2+]i, which could be prevented by pretreatment of the cells with either TPA or W-7. Our data suggest that pertussis toxin-sensitive G protein(s) are involved in ATP-dependent activation of PI turnover and in secretion of surfactant in type II cells. Activation of protein kinase C blocks the ATP-stimulated increase in [Ca2+]i. Finally, calmodulin may be involved in the regulation of ATP-dependent increase in [Ca2+]i, the activation of PI turnover, and the secretion of surfactant in type II cells. lung; exocytosis; cell calcium; G proteins; phosphoinositide turnover

scholarly journals P2-purinoceptor regulation of surfactant phosphatidylcholine secretion. Relative roles of calcium and protein kinase C

1990 ◽  
Vol 266 (2) ◽  
pp. 407-413 ◽  
Author(s):  
W R Rice ◽  
C C Dorn ◽  
F M Singleton
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Kinase C ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Phosphatidylcholine Secretion

Ca2+ and protein kinase C have both been proposed as intracellular signals for subsequent phosphatidylcholine secretion by alveolar Type II cells. We have determined the relative roles of Ca2+ and protein kinase C in regulating surfactant phosphatidylcholine secretion by utilizing exogenous ATP and the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) as secretagogues, along with MAPTAM to chelate intracellular Ca2+ and sphingosine to inhibit endogenous protein kinase C. Exposure of Type II cells to the P2-purinoceptor agonist, ATP, results in a dose-dependent increase in surfactant phosphatidylcholine secretion from isolated alveolar Type II cells with an EC50 (concn. producing 50% of maximal response) of 2 microM. Administration of exogenous ATP to Type II cells also results in a dose-dependent increase in inositol trisphosphate production, Ca2+ mobilization and [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding as a measure of protein kinase C translocation. The EC50 in each case is 1-5 microM, indicating association of these events with surfactant phosphatidylcholine secretion. Loading Type II cells with non-hydrolysable GTP analogue (GTP[S]) inhibited ATP-induced Ca2+ mobilization, supporting the hypothesis that Type II cell P2-purinoceptors are coupled to phospholipase C via a GTP-binding protein. The ATP-induced elevation of cytosolic Ca2+ was also inhibited by MAPTAM (a cell-permeant EGTA analogue) by 90%, but MAPTAM was without effect on surfactant phosphatidylcholine secretion induced by ATP. Sphingosine inhibited both ATP- and TPA-induced surfactant phosphatidylcholine secretion as well as [3H]PDBu binding with a similar IC50 (concn. producing 50% of maximal inhibition) (10 microM). Sphingosine did not affect surfactant phosphatidylcholine secretion induced by terbutaline and did not have a significant effect on Ca2+ mobilization induced by exogenous ATP. These results are consistent with a prominent role for protein kinase C in regulation of P2-purinoceptor-induced surfactant phosphatidylcholine secretion, and indicate that Ca2+ mobilization is not a necessary step for ATP-induced surfactant phosphatidylcholine secretion.

Protein kinase C in intracellular pH regulation in alveolar type II cells

1996 ◽  
Vol 271 (1) ◽  
pp. L106-L113 ◽  
Author(s):  
S. Wadsworth ◽  
A. M. Wu ◽  
A. Spitzer ◽  
A. Chander
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Ph ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Dependent Manner ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Kinase C ◽  
Acid Load

The Na+/H+ exchanger and Na(+)-HCO3- cotransporter have been implicated in regulation of intracellular pH (pHi) in alveolar type II cells. This study demonstrates that activation of protein kinase C (PKC) stimulates both of these ion transporters in type II cells. Treatment of type II cells with 80 nM phorbol 12-myristate 13-acetate (PMA) increased the resting pHi in a time-dependent manner. Compared with control cells, the rates of recovery from an acid load increased with PMA treatment, reaching a maximum at 15 min, and returned to control levels by 3 h. The PMA-stimulated changes in recovery rate were sensitive to H-7, a PKC inhibitor. For PMA treatment up to 2 h, these recoveries were also sensitive to dimethylamiloride (DMA), an inhibitor of Na+/H+ exchanger activity, and to HCO3-, suggesting activation of both the Na+/H+ exchanger and the Na(+)-HCO3- cotransporter. After prolonged (3 h) treatment with PMA, however, the recovery was insensitive to DMA but was sensitive to HCO3-, suggesting that the Na+/H+ exchanger was no longer active and that most of the recovery was mediated by the Na(+)-HCO3- cotransporter. PMA treatment also altered the Na+ kinetics of the recovery from an acid load with respect to the Michaelis constant (Km) and maximal ion flux (Vmax), suggesting protein modifications of each transporter. We suggest that PKC activation in type II cells results in acute and long-term changes in pHi regulatory mechanisms mediated by the Na+/H+ exchanger and by the Na(+)-HCO3- cotransporter.

Osmotic stress induces both secretion and apoptosis in rat alveolar type II cells

1998 ◽  
Vol 275 (4) ◽  
pp. L670-L678 ◽  
Author(s):  
Yasmin S. Edwards ◽  
Leanne M. Sutherland ◽  
John H. T. Power ◽  
Terence E. Nicholas ◽  
Andrew W. Murray
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Osmotic Shock ◽  
Primary Cultures ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Kinase C ◽  
Extracellular Signal

The aim of this study was to analyze the effects of osmotic shock and secretagogues such as ATP and 12- O-tetradecanoylphorbol 13-acetate (TPA) on various intracellular signaling pathways in primary cultures of alveolar type II cells and examine their potential role in regulating events such as secretion and apoptosis in these cells. Sorbitol-induced osmotic stress caused the sustained release of [3H]phosphatidylcholine ([3H]PC) from primary cultures of rat alveolar type II cells prelabeled with [3H]choline chloride. This release was not dependent on protein kinase C because downregulation of the major protein kinase C isoforms (α, βII, δ, and η) expressed in alveolar type II cells had no effect on [3H]PC secretion. Sorbitol, as well as the known secretagogues TPA and ATP, activated extracellular signal-regulated kinase. Although an inhibitor of the extracellular signal-regulated kinase cascade, PD-98059, blocked this activation, it had no effect on the release of [3H]PC. Sorbitol and ultraviolet C radiation, but not TPA or ATP, were also found to activate both p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Furthermore, both sorbitol and ultraviolet C radiation induced apoptosis in alveolar type II cells as demonstrated by Hoechst 33258 staining of the condensed nuclei, the generation of DNA ladders, and the activation of caspases. The data indicate that multiple signaling pathways are activated by traditional secretagogues such as TPA and ATP and by cellular stresses such as osmotic shock and that these may be involved in regulating secretory and apoptotic events in alveolar type II cells.

Interaction between β-adrenergic signaling and protein kinase C increases cytoplasmic Ca2+ in alveolar type II cells

Life Sciences ◽  
2001 ◽  
Vol 68 (21) ◽  
pp. 2361-2371 ◽  
Author(s):  
Yoichiro Isohama ◽  
Miyuki Kanemaru ◽  
Hirofumi Kai ◽  
Kazuo Takahama ◽  
Takeshi Miyata
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Adrenergic Signaling ◽  
Kinase C

Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells

1991 ◽  
Vol 261 (4) ◽  
pp. 105-109 ◽  
Author(s):  
T. A. Voyno-Yasenetskaya ◽  
L. G. Dobbs ◽  
M. C. Williams
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Pertussis Toxin ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Calmodulin Antagonist ◽  
Pi Turnover ◽  
Kinase C ◽  
Surfactant Secretion

Several different classes of agonists are known to stimulate exocytosis in type II cells. These agonists cause increases in second messengers, such as adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+, and/or stimulate protein kinase C. We studied generation of cAMP and phosphoinositide (PI) turnover in monolayer cultures of type II cells and measured [Ca2+]i in single cultured cells. ATP [10–4 M], which stimulates secretion of phosphatidylcholine (PC) and increases cellular cAMP, also stimulated PI turnover and increased [Ca2+]i. 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulates PC secretion and activates protein kinase C, did not increase [Ca2+]i. Pretreatment of type II cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the PC secretion induced by ATP and TPA and blocked the increase in PI turnover caused by ATP. ATP-dependent surfactant secretion and stimulation of PI turnover could also be inhibited by pretreatment of the cells with pertussis toxin. We used the fluorescent probe indo-1 to measure [Ca2+]i in single cultured type II cells. ATP produced rapid transient increases in [Ca2+]i, which could be prevented by pretreatment of the cells with either TPA or W-7. Our data suggest that pertussis toxin-sensitive G protein(s) are involved in ATP-dependent activation of PI turnover and in secretion of surfactant in type II cells. Activation of protein kinase C blocks the ATP-stimulated increase in [Ca2+]i. Finally, calmodulin may be involved in the regulation of ATP-dependent increase in [Ca2+]i, the activation of PI turnover, and the secretion of surfactant in type II cells. lung; exocytosis; cell calcium; G proteins; phosphoinositide turnover

Protein kinase C in ATP regulation of lung surfactant secretion in type II cells

1995 ◽  
Vol 268 (1) ◽  
pp. L108-L116 ◽  
Author(s):  
A. Chander ◽  
N. Sen ◽  
A. M. Wu ◽  
A. R. Spitzer
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Indirect Evidence ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Dependent Manner ◽  
Type Ii ◽  
Kinase C ◽  
Surfactant Secretion

Previous studies provided indirect evidence for a role for protein kinase C (PKC) in ATP stimulation of surfactant secretion. The present study demonstrates that ATP increases PKC activity in the membrane fraction and decreases PKC activity in the cytosol fraction of alveolar type II cells, indicating translocation of PKC to the membranes. The kinetics of ATP concentration dependence of increases in phosphatidylcholine secretion and diacylglycerol content were similar, suggesting direct correlation between these two parameters. ATP also increased membrane PKC activity in a concentration-dependent manner. Almost one-half of the PKC activity in the cytosol and membrane fractions was Ca2+ independent. The ATP-induced increase was greater in membrane-associated Ca(2+)-dependent enzyme (233%) than in Ca(2+)-independent enzyme (121%). Desensitization of PKC by exposure of cells to phorbol esters decreased PKC activity in the membrane and cytosol fractions. In cells pretreated for 3 h with phorbol esters, PKC activity was near minimum, and ATP-stimulated secretion was lowest (; 40% of that observed in untreated cells). These results indicate that a major part of ATP-stimulated surfactant secretion in type II cells is mediated via activation of PKC.

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