thapsigargin and phorbol-12-13-didecanoate

In acute respiratory distress syndrome, pulmonary vascular permeability increases, causing intravascular fluid and protein to move into the lung's interstitium. The classic model describing the formation of pulmonary edema suggests that fluid crossing the capillary endothelium is drawn by negative interstitial pressure into the potential space surrounding extra-alveolar vessels and, as interstitial pressure builds, is forced into the alveolar air space. However, the validity of this model is challenged by animal models of acute lung injury in which extra-alveolar vessels are more permeable than capillaries under a variety of conditions. In the current study, we sought to determine whether extravascular fluid accumulation can be produced because of increased permeability of either the capillary or extra-alveolar endothelium, and whether different pathophysiology results from such site-specific increases in permeability.. We perfused isolated lungs with either the plant alkaloid thapsigargin, which increases extra-alveolar endothelial permeability, or with 4alpha-phorbol 12, 13-didecanoate, which increases capillary endothelial permeability.. Both treatments produced equal increases in whole lung vascular permeability, but caused fluid accumulations in separate anatomical compartments. Light microscopy of isolated lungs showed that thapsigargin caused fluid cuffing of large vessels, while 4alpha-phorbol 12, 13-didecanoate caused alveolar flooding. Dynamic compliance was reduced in lungs with cuffing of large vessels, but not in lungs with alveolar flooding.. Phenotypic differences between vascular segments resulted in site-specific increases in permeability, which have different pathophysiological outcomes. Our findings suggest that insults leading to acute respiratory distress syndrome may increase permeability in extra-alveolar or capillary vascular segments, resulting in different pathophysiological sequela.

Topics: Animals; Carcinogens; Cell Membrane Permeability; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Extravascular Lung Water; Lung; Lung Compliance; Male; Phorbol Esters; Pulmonary Alveoli; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Respiratory Mechanics; Thapsigargin

Sphingosine-1-phosphate, a metabolite of sphingolipids which has previously been shown to stimulate DNA synthesis and cell division in quiescent cultures of Swiss 3T3 fibroblasts (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167), induced a transient increase in intracellular free calcium independent of extracellular calcium. The increase in calcium was completely abolished when intracellular calcium pools were depleted with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. The dose-response for calcium release induced by sphingosine-1-phosphate correlated closely with the concentration required for stimulation of DNA synthesis. The magnitude of the calcium response decreased with successive challenges, although sphingosine-1-phosphate did not attenuate the responses to either bradykinin or ionomycin. Conversely, prior stimulation of the cells with bradykinin had no effect on the sphingosine-1-phosphate-induced calcium signal. Although sphingosine-1-phosphate increased inositol (1,4,5)-trisphosphate levels, complete inhibition of inositol phosphate formation by pretreatment with 12-O-tetradecanoylphorbol-13-acetate did not block sphingosine-1-phosphate-mediated calcium responses. Moreover, in permeabilized cells, heparin, an inositol (1,4,5)-trisphosphate antagonist, blocked Ca2+ release induced by inositol (1,4,5)-trisphosphate, but did not significantly alter the Ca2+ release induced by sphingosine-1-phosphate. Sphingosine-1-phosphate did not stimulate the release of arachidonic acid, another signaling molecule known to elevate [Ca2+]i without inositol lipid turnover or calcium influx. Our data suggest that sphingosine-1-phosphate mobilizes Ca2+ from internal stores primarily through a mechanism independent of inositol lipid hydrolysis and arachidonic acid release and that sphingolipid metabolism may be important in calcium homeostasis.

Topics: 3T3 Cells; Animals; Arachidonic Acid; Bradykinin; Calcium; Calcium-Transporting ATPases; Cytosol; DNA; Egtazic Acid; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionomycin; Kinetics; Lysophospholipids; Mice; Phorbol Esters; Second Messenger Systems; Sphingosine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Time Factors

Calcyclin is a member of the S-100 family of calcium-binding proteins, whose expression is enhanced when quiescent cells are exposed to mitogenic signals. The function of calcyclin is unknown, but it is thought to be involved in modulating the intracellular calcium concentration following mitogenic stimuli. Since activation of protein kinase C (PKC) also occurs following stimulation of quiescent cells by a variety of mitogens, we have investigated the relationship between calcyclin expression and PKC activation in three human endometrial adenocarcinoma cell lines. The addition of 10(-7) M 4 beta-phorbol 12-myristate 13-acetate (PMA) to HEC-50 and HEC-1B cell cultures resulted in a change in cell morphology, an inhibition of proliferation, an increase in calcyclin transcription rate, and an increase in calcyclin mRNA and calcyclin protein levels. In contrast, PMA had no effect on cell morphology or cell proliferation in the Ishikawa adenocarcinoma cell line but enhanced calcyclin expression. Another bioactive phorbol ester had the same effect, whereas the calcium ionophore A23187 and the non-phorbol-ester-type tumor promoter thapsigargin had no effect on calcyclin expression. The effect of PMA on calcyclin expression was blocked by the simultaneous addition of the PKC inhibitor staurosporine and by protein synthesis inhibition with cycloheximide. RNase protection assays and primer extension analysis demonstrated that PMA enhanced transcription from all three of the previously identified transcription start sites in the calcyclin gene. These data clearly demonstrate a dissociation between calcyclin expression and cellular proliferation and suggest that the enhanced calcyclin expression which is seen in quiescent cells following mitogenic stimuli may result from activation of the PKC system.

Topics: Adenocarcinoma; Alkaloids; Calcimycin; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Division; Cycloheximide; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; RNA, Messenger; RNA, Neoplasm; S100 Calcium Binding Protein A6; S100 Proteins; Signal Transduction; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Uterine Neoplasms