Regulation of secretion in Clara cells: studies using the isolated perfused rat lung.

Previous studies from our laboratory indicated that both beta-adrenergic and cholinergic agents stimulate in vivo secretion by rat bronchiolar Clara cells. Those studies also provided support for an in-series beta-adrenergic-cholinergic stimulation of secretion. To further explore the regulation of secretion in Clara cells, and to do it in the absence of systemic influences, we have used the isolated ventilated perfused rat lung. We have again used morphometry and electron microscopy to assess secretion by measuring the volume density (fraction of cell volume) of the secretory granules of bronchiolar Clara cells. We found that in the isolated perfused lung, as in the intact animal, isoproterenol stimulated secretion in Clara cells and that this effect was blocked by the beta-adrenergic antagonist propranolol. Pilocarpine, unlike its action in the intact animal, did not stimulate secretion in the isolated lung; rather it inhibited the secretory effect of isoproterenol. Increased tidal-volume ventilation stimulated secretion; propranolol did not block this effect. Analogs of cyclic (c)AMP and of cGMP also stimulated secretion by Clara cells. These findings indicate that there are at least two mechanisms by which Clara cells can be stimulated to secrete. One seems to be beta-adrenergic-cAMP mediated but the triggering event is unknown. The other is initiated by increased tidal volume and cGMP may be involved in the intracellular mediation of this stimulatory event. Finally, we found evidence of beta-adrenergic (stimulatory) -cholinergic (inhibitory antagonism in the regulation of secretion in Clara cells.

Inhibition of human polymorphonuclear leukocyte function by 2-cyclohexene-1-one. A role for glutathione in cell activation.

2-cyclohexene-1-one and diethyl maleate specifically decrease reduced glutathione (GSH) levels in human polymorphonuclear leukocytes (PMN) by direct conjugation, and by interaction with the glutathione-s-transferase system. Using these two nontoxic reagents we have examined the effect of decreased GSH levels on five parameters of PMN activation: superoxide generation, release of the lysosomal enzymes lysozyme and beta-glucuronidase, and increases in the influx of Na+ and Ca2+. When PMN pretreated with 2-cyclohexene-1-one or diethyl maleate were incubated with formyl-methionyl-leucyl-phenylalanine (FMLP) or the proteolytic fragment of the fifth component membrane of complement, C5a, agents that interact with surface membrane receptors, increases in all five parameters were inhibited in a dose-dependent manner. For O-2 generation and lysosomal enzyme release the ID50 for 2-CHX-1 was 40--90 micrometers corresponding with a 30--50% decrease in intracellular GHS. In contrast stimulation of treated PMN by the divalent cation ionophore A23187 or 5-hydroxyeicosatetraenoic acid was much less sensitive to depressed GSH; the ID50 for 2-cyclohexene-1-one was 1 mM or greater, corresponding with an 80--90% decrease in GSH. The effect of lowered GSH was not the result of decreased binding of FMLP to surface receptors because [3H]-FMLP binding studies demonstrated a two- to three-fold increase in the number of available binding sites. These data indicate that normal GSH levels are necessary for the transduction of the activation signal from the exterior to the interior of the PMN, but once initiated the activation sequence proceeds normally despite markedly lowered intracellular GSH.

Suicidal hanging. An association with the adult respiratory distress syndrome.

This study of two victims of suicidal hanging describes a previously unknown association between near-fatal hanging and the adult respiratory distress syndrome. We report on the pathophysiologic results of this pulmonary complication and the implications of this finding regarding the treatment of these patients.

Relationship of biosynthesis of slow reacting substance to intracellular glutathione concentrations.

To further elucidate the role of glutathione (GSH) in the biosynthesis of slow reacting substance (SRS), SRS generation was studied in rat basophilic leukemia cells that had been preincubated with 2-cyclohexen-1-one or diethyl maleate to decrease their intracellular GSH concentrations. At low GSH levels SRS formation was markedly inhibited. The formation of other lipoxygenase products was much less affected, although some decrease in 5-hydroxyicosatetraenoic acid formation also occurred, apparently due in part to less rapid reduction of the 5-hydroperoxide.

Inhibition of lectin-induced lymphocyte activation by 2-cyclohexene-1-one: analysis of DNA synthesis in individual cells by BUdR quenching of Hoechst 33258.

A novel technique utilizing the quenching of fluorescence Hoechst 32258 by bromodeoxyuridine (BUdR) was used to investigate the effect of depressed glutathione (GSH) on the activation of human peripheral blood lymphocytes by phytohemagglutinin (PHA) or concanavalin A (con A). This technique allows the quantification of DNA synthesis in individual cells. Lymphocytes were purified by Ficoll-Hypaque density gradient centrifugation and treated with 5 X 10(-5) M to 1 X 10(-6) M 2-cyclohexene-1-one (2-CHX-1), a reagent which specifically depletes intracellular GSH, and/or interferes with GSH-protein interactions, and 25 micrograms/ml BUdR in the presence or absence of PHA or con A. At 72 h lymphocyte smears were stained with Hoechst 33258 and examined using a computer controlled microscope photometer. When DNA synthesis was assayed using BUdR quenching two populations of lymphocytes were noted; a population which incorporated little or no BUdR (unactivated) and a population which incorporated BUdR sufficient to quench 33258 fluorescence by approximately 35%. Cells treated with graded doses of 2-CHX-1 which reduced glutathione levels by 10-90%, showed a progressive loss of cells from the activated population and the appearance of these cells in the inactivated population. Statistical analysis of the frequency histograms demonstrated that there were no cells which incorporated an intermediate amount of BUdR. This data demonstrates that depressed intracellular GSH or inhibition of GSH-protein interactions inhibits an early step in the biochemical sequence of events leading to DNA synthesis but does not inhibit the DNA synthetic process per se.

Inhibition of lectin-induced lymphocyte activation by 2-cyclohexene-1-one: decreased intracellular glutathione inhibits an early event in the activation sequence.

The importance of normal intracellular glutathione (GSH) levels in the activation of human peripheral blood lymphocytes (PBL) by mitogenic lectins was explored using 2-cyclohexene-1-one (2-CHX-1), an agent that selectively decreases the levels of intracellular GSH. PBL incubated with mitogenic lectins and graded doses of 2-CHX-1 showed a dose-dependent inhibition of activation assayed by [3H]-thymidine uptake and percentage of blast transformation. 2.5 x 10(-5) M 2-CHX-1, a concentration that decreases GSH levels to less than 20% of control, caused an 80 to 90% suppression of both [3H]-thymidine uptake and blast transformation; lower concentrations were less effective. Time course studies showed that 2-CHX-1 was maximally effective only if added during the first 4 hr of culture. At the concentrations used, 2-CHX-1 was not cytotoxic to lectin-stimulated or unstimulated PBL and did not interfere with mitogen-lymphocyte interaction. These results suggest that adequate levels of glutathione are necessary for lymphocyte activation and that the glutathione requirement is exerted in the early activation sequence.